Antibodies that bind both il-17a and il-17f and methods of using the same

ABSTRACT

The present invention relates to blocking, inhibiting, reducing, antagonizing or neutralizing the activity of IL-17A and IL-17F. IL-17A and IL-17F are cytokines that are involved in inflammatory processes and human disease. The present invention includes antibodies that bind both IL-17A and IL-17F, as well as methods of using the same in inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/364,810, filed Feb. 2, 2012, which is a continuation of U.S.patent application Ser. No. 13/198,933, filed Aug. 5, 2011, nowabandoned, which is a continuation of U.S. patent application Ser. No.12/617,078, filed Nov. 12, 2009, now abandoned, which is a continuationof U.S. patent application Ser. No. 11/684,907, filed Mar. 12, 2007, nowabandoned, which claims the benefit of U.S. Patent Application Ser. No.60/781,121, filed Mar. 10, 2006, U.S. Patent Application Ser. No.60/828,271, filed Oct. 5, 2006, and U.S. Patent Application Ser. No.60/862,501, filed Oct. 23, 2006, all of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of antibodies that bind to both IL-17A and IL-17F and methodsof using the same.

BACKGROUND OF THE INVENTION

Six members of the IL-17 family have been identified based on theirsimilarity to the prototypical member of the family, originallyidentified as IL-17 and which is now designated IL-17A. See e.g.Spriggs, M. K. “Interleukin-17 and its receptor” J. Clin. Immunol.17:366-369 (1997). The other members of the family are IL-17, IL-17C,IL-17D, IL-17E (also known as IL-25), and IL-17F. See e.g. Kawaguchi etal. “IL-17 cytokine family,” J. Allergy Clin. Immunol. 114: 1265-1273(2004); Kolls and Linden, “Interleukin-17 family members andinflammation,” Immunity 21:467-476 (2004) and Moseley et al.,“Interleukin-17 family and IL-17 receptors,” Cytokine Growth Factor Rev14:155-174 (2003). Among the members of the family, IL-17A and IL-17Fare by far the most similar to one another sharing 55% identity (Kollsand Linden, 2004). In addition to their sequence similarity, both ofthese cytokines seem are produced by similar cell types, most notablyactivated, memory CD4+ T cells. See e.g. Agarwal et al., “Interleukin-23promotes a distinct CD4 T cell activation state characterized by theproduction of interleukin-17” J. Biol. Chem. 278:1910-191 (2003); seealso Langrish et al. “IL-23 drives a pathogenic T cell population thatinduces autoimmune inflammation” J. Exp. Med. 201: 233-240 (2005); andStarnes et al. “Cutting edge: IL-17F, a novel cytokine selectivelyexpressed in activated T cells and monocytes, regulates angiogenesis andendothelial cell cytokine production” J. Immunol. 167:4137-4140 (2001).

Moreover, both have been similarly implicated as contributing agents toprogression and pathology of a variety of inflammatory and auto-immunediseases in humans and in mouse models of human diseases. Specifically,IL-17A and IL-17F have been implicated as major effector cytokines thattrigger inflammatory responses and thereby contribute to a number ofautoinflammatory diseases including multiple sclerosis, rheumatoidarthritis, and inflammatory bowel diseases.

The demonstrated in vivo activities of both IL-17A and IL-17F illustratethe clinical or therapeutic potential of, and need for, IL-17A andIL-17F antagonists. Specifically, antibodies that bound to both IL-17Aand IL-17F that inhibit (antagonist antibodies) the immunologicalactivities of both IL-17A and Il-17F would possess such noveltherapeutic qualities. Thus, there remains a need in the art for anantagonist to both IL-17A and IL-17F.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE shows a representative binding curve generated by the Prizmsoftware program.

DETAILED DESCRIPTION OF THE INVENTION

The pro-inflammatory cytokines IL-17A and IL-17F have a high degree ofsequence similarity, share many biological properties, and are bothproduced by activated T cells. They have both been implicated as factorsthat contribute to the progression of various autoimmune andinflammatory diseases including rheumatoid arthritis and asthma. Infact, reagents that negate IL-17A function significantly amelioratedisease incidence and severity in several mouse models of human disease.IL-17A mediates its effects through interaction with its cognatereceptor, the IL-17 receptor (IL-17RA), and for IL-17F, IL-17RA. Thus,the present invention has determined that a cross-reactive antibody maybe useful as an antagonist to both IL-17A and IL-17F, and thus to blockboth IL-17A and IL-17F. Accordingly, the present invention addressesthis need by providing therapeutic molecules (e.g. antibodies) which mayblock, inhibit, reduce, antagonize or neutralize the activity of bothIL-17A (polynucleotide sequence is shown as SEQ ID NO:1 and the encodedpolypeptide is shown as SEQ ID NO:2) and IL-17F (polynucleotide sequenceis shown as SEQ ID NO:3 and the encoded polypeptide is shown as SEQ IDNO:4). Thus, the present invention is directed to IL-17A and IL-17Fantagonists, such as the antibodies described herein. The inventionfurther provides uses therefor in inflammatory disease, as well asrelated compositions and methods.

A) OVERVIEW

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multi-step pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), psoriasis, and asthma), non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

A central event in both humoral and cell mediated immune responses isthe activation and clonal expansion of helper T cells. Helper T cellactivation is initiated by the interaction of the T cell receptor(TCR)-CD3 complex with an antigen-MHC on the surface of an antigenpresenting cell. This interaction mediates a cascade of biochemicalevents that induce the resting helper T cell to enter a cell cycle (theG0 to G1 transition) and results in the expression of a high affinityreceptor for IL-2 and sometimes IL-4. The activated T cell progressesthrough the cycle proliferating and differentiating into memory cells oreffector cells.

In addition to the signals mediated through the TCR, activation of Tcells involves additional costimulation induced by cytokines released bythe antigen presenting cell or through interactions with membrane boundmolecules on the antigen presenting cell and the T cell. The cytokinesIL-1 and IL-6 have been shown to provide a costimulatory signal. Also,the interaction between the B7 molecule expressed on the surface of anantigen presenting cell and CD28 and CTLA-4 molecules expressed on the Tcell surface effect T cell activation. Activated T cells express anincreased number of cellular adhesion molecules, such as ICAM-1,integrins, VLA-4, LFA-1, CD56, etc.

T-cell proliferation in a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In many immune responses, inflammatorycells infiltrate the site of injury or infection. The migrating cellsmay be neutrophilic, eosinophilic, monocytic or lymphocytic as can bedetermined by histologic examination of the affected tissues. CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons,Inc.

Immune related diseases could be treated by suppressing the immuneresponse. Using soluble receptors and/or neutralizing antibodies thatinhibit molecules having immune stimulatory activity would be beneficialin the treatment of immune-mediated and inflammatory diseases. Moleculeswhich inhibit the immune response can be utilized (proteins directly orvia the use of antibody agonists) to inhibit the immune response andthus ameliorate immune related disease.

Interleukin-17 (IL-17A) has been identified as a cellular ortholog of aprotein encoded by the T lymphotropic Herpes virus Saimiri (HSV) [see,Rouvier et al., J. Immunol., 150(12): 5445-5456 (19993); Yao et al., J.Immunol., 122(12):5483-5486 (1995) and Yao et al., Immunity,3(6):811-821 (1995)]. Subsequent characterization has shown that thisprotein is a potent cytokine that acts to induce proinflammatoryresponses in a wide variety of peripheral tissues. IL-17A is adisulfide-linked homodimeric cytokine of about 32 kDa which issynthesized and secreted only by CD4+ activated memory T cells (reviewedin Fossiez et al., Int. Rev. Immunol., 16: 541-551 [1998]).Specifically, IL-17 is synthesized as a precursor polypeptide of 155amino acids with an N-terminal signal sequence of 19-23 residues and issecreted as a disulfide-linked homodimeric glycoprotein. Il-17A isdisclosed in WO9518826 (1995), WO9715320 (1997) and WO9704097 (1997), aswell as U.S. Pat. No. 6,063,372.

Despite its restricted tissue distribution, IL-17A exhibits pleitropicbiological activities on various types of cells. IL-17A has been foundto stimulate the production of many cytokines. It induces the secretionof IL-6, IL-8, IL-12, leukemia inhibitory factor (LIF), prostaglandinE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes,epithelial and endothelial cells. IL-17A also has the ability to induceICAM-1 surface expression, proliferation of T cells, and growth anddifferentiation of CD34⁺ human progenitors into neutrophils. IL-17A hasalso been implicated in bone metabolism, and has been suggested to playan important role in pathological conditions characterized by thepresence of activated T cells and TNF-α production such as rheumatoidarthritis and loosening of bone implants (Van Bezooijen et al., J. BoneMiner. Res. 14: 1513-1521 [1999]). Activated T cells of synovial tissuederived from rheumatoid arthritis patients were found to secrete higheramounts of IL-17A than those derived from normal individuals orosteoarthritis patients (Chabaud et al., Arthritis Rheum. 42: 963-970[1999]). It was suggested that this proinflammatory cytokine activelycontributes to synovial inflammation in rheumatoid arthritis. Apart fromits proinflammatory role, IL-17A seems to contribute to the pathology ofrheumatoid arthritis by yet another mechanism. For example, IL-17A hasbeen shown to induce the expression of osteoclast differentiation factor(ODF) mRNA in osteoblasts (Kotake et al., J. Clin. Invest., 103:1345-1352 [1999]). ODF stimulates differentiation of progenitor cellsinto osteoclasts, the cells involved in bone resorption.

Since the level of IL-17A is significantly increased in synovial fluidof rheumatoid arthritis patients, it appears that IL-17A inducedosteoclast formation plays a crucial role in bone resorption inrheumatoid arthritis. IL-17A is also believed to play a key role incertain other autoimmune disorders such as multiple sclerosis(Matusevicius et al., Mult. Scler., 5: 101-104 [1999]). IL-17A hasfurther been shown, by intracellular signalling, to stimulate Ca.sup.2+influx and a reduction in [cAMP], in human macrophages (Jovanovic etal., J. Immunol., 160:3513 [1998]). Fibroblasts treated with IL-17Ainduce the activation of NF-κB, [Yao et al., Immunity, 3:811 (1995),Jovanovic et al., supra], while macrophages treated with it activateNF-κB and mitogen-activated protein kinases (Shalom-Barek et al., J.Biol. Chem., 273:27467 [1998]).

Additionally, IL-17A also shares sequence similarity with mammaliancytokine-like factor 7 that is involved in bone and cartilage growth.Other proteins with which IL-17A polypeptides share sequence similarityare human embryo-derived interleukin-related factor (EDIRF) andinterleukin-20.

Consistent with IL-17A's wide-range of effects, the cell surfacereceptor for IL-17A has been found to be widely expressed in manytissues and cell types (Yao et al., Cytokine, 9:794 [1997]). While theamino acid sequence of the human IL-17A receptor (IL-17RA) (866 aminoacids) predicts a protein with a single transmembrane domain and a long,525 amino acid intracellular domain, the receptor sequence is unique andis not similar to that of any of the receptors from the cytokine/growthfactor receptor family. This coupled with the lack of similarity ofIL-17A itself to other known proteins indicates that IL-17A and itsreceptor may be part of a novel family of signalling proteins andreceptors. It has been demonstrated that IL-17A activity is mediatedthrough binding to its unique cell surface receptor, wherein previousstudies have shown that contacting T cells with a soluble form of theIL-17A receptor polypeptide inhibited T cell proliferation and IL-2production induced by PHA, concanavalin A and anti-TCR monoclonalantibody (Yao et al., J. Immunol., 155:5483-5486 [1995]). As such, thereis significant interest in identifying and characterizing novelpolypeptides having homology to the known cytokine receptors,specifically IL-17A receptors.

The expression pattern of IL-17F appears to be similar to that ofIL-17A, such that it includes only activated CD4+ T cells and monocytes(Starnes et al. J. Immunol. 167: 4137-4140 [2001]). IL-17F has beendemonstrated to induce G-CSF, IL-6, and IL-8 in fibroblasts (Hymowitz etal, EMBO J. 20:5322-5341 [2001]) and TGF-b in endothelial cells (Starneset al. J. Immunol. 167: 4137-4140 [2001]). It has recently been reportedthat IL-23, a cytokine produced by dendritic cell, can mediate theproduction of both IL-17A and IL-17F, primarily in memory T cells(Aggarwal et al. J. Biol. Chem. 278:1910-1914 [2003]).

Moreover, over expression or upregulation of both IL-17A and IL-17F havebeen shown in arthritic and asthmatic individuals (reviewed in Moseleyet al. CytokineGrowth Factor Rev 14:155-174 [2003]). With regards toarthritis, these cytokines act in a manner characteristic to thecartilage and joint destruction that is associated with rheumatoid- andosteo-arthritis. For example, IL-17A and IL-17F have been demonstratedto enhance matrix degradation in articular cartilage explants viarelease of cartilage proteoglycan glycosaminoglycans and collagenfragments, while inhibiting the synthesis of new proteoglycans andcollagens (Cai et al. Cytokine 16:10-21 [2001]; Attur et al ArthritisRheum 44:2078-2083 [2001]).

Similar to IL-17A, overexpression of IL-17F in mice has also been shownto increase lung neutrophil recruitment and result in increasedexpression of Th1-associated cytokines in the lung, including IL-6,IFN-gamma, IP-10 and MIG (Starnes et al. J. Immunol. 167: 4137-4140[2001]). IL-17F was also upregulated in T cells from allergen-challengedasthmatics (Kawaguchi et al J. Immunol 167:4430-4435 [2001]), and foundto induce IL-6 and IL-8 production in NHBE. In contrast to IL-17A,IL-17F appears to inhibit angiogenesis in vitro (Starnes et al. J.Immunol. 167: 4137-4140 [2001]).

IL-17F mRNA was not detected by northern blot in various human tissuesbut was dramatically induced upon activation of CD4+ T cells andmonocytes. Id. In mice, Th2 cells and mast cells were found to expressIL-17F upon activation. See Dumont, Expert Opin. Ther. Patents 13(3)(2003). Like IL-17A, the expression of IL-17F was also found to beupregulated by IL-23 in mouse.

The IL-17 cytokine/receptor families appear to represent a uniquesignaling system within the cytokine network that will offer innovativeapproaches to the manipulation of immune and inflammatory responses.Accordingly, the present invention is directed to antibodies that bindto both IL-17A and IL-17F.

The present invention provides antibodies that bind both IL-17A andIL-17F (IL-17A/F antibodies) and methods for using IL-17A/F antibodies.The antibodies may act as antagonists or agonists, and find utility for,among other things, in vitro, in situ, or in vivo diagnosis or treatmentof mammalian cells or pathological conditions associated with thepresence (or absence) of IL-17A and/or IL-17F.

Preferred embodiments of the invention include antibodies, and anyfragments or permutations thereof that bind to both IL-17A and IL-17F(herein refereed to interchangeably as “cross-reactive antibodies,” “A/Fantibodies,” “bispecific antibodies,” “IL-17A/F antibodies,” etc.).Specifically, such antibodies are capable of specifically binding toboth human IL-17A and IL-17F and/or are capable of modulating biologicalactivities associated with either or both IL-17A and IL-17F and/or theirreceptors, IL-17RA and IL-17RC, and thus are useful in the treatment ofvarious diseases and pathological conditions such as immune relateddiseases. In more particular embodiments, there are provided antibodieswhich specifically bind to IL-17A (SEQ ID NO:2) and IL-17F (SEQ IDNO:4). Optionally, the antibody is a monoclonal antibody.

For example, the IL-17A/F antibodies bind to an epitope on both IL-17Aand IL-17F, wherein said epitope comprises residues Ile(23), Lys (25),Gly(27), Thr (29) and Pro(34) of the following sequences of human IL-17Fand the equivalent sequence found in human IL-17A shown below. Residues23, 25, 27, 29, and 34 are predicted to be on the surface of both IL-17Aand IL-17F and therefore are accessible to the binding of an antibody ofthe present invention or an equivalent protein binding antagonist.

(Ile23-Pro34 of SEQ ID NO: 4) hIL17F IPKVGHTFFQKP(Ile20-Pro31 of SEQ ID NO: 2) hIL17A IVKAGITIPRNP

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope comprises residues Arg(67),Ser(68), Thr(69), Ser(70), Pro(71), Trp(72), Asn(73) of the followingsequences of human IL-17F and the equivalent sequence found in humanIL-17A, as shown below. Residues 69, 71 and 73 are predicted to be onthe surface of the bioactive cytokine and therefore are accessible tothe binding of an antibody of the present invention or equivalentprotein binding antagonist.

(Arg67-Asn73 of SEQ ID NO: 4) hIL17F RSTSPWN(Arg69-Asn75 of SEQ ID NO: 2) hIL17A RSTSPWN

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope comprises residues Asp(79),Pro(80), Asn(81), Arg(82), Tyr(83), Pro(84) and Ser(85) of the followingsequences of human IL-17F and the equivalent sequence found in humanIL-17A, as shown below. All residues of this epitope are predicted to beon the surface of the bioactive cytokine and therefore are accessible tothe binding of an antibody of the present invention or equivalentprotein binding antagonist.

(Asp79-Ser85 of SEQ ID NO: 4) hIL-17F DPNRYPS(Asp81-Ser87 of SEQ ID NO: 2) hIL-17A DPERYPS

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope comprises residues Thr(146),Pro(147), Val(148), Ile(149), His(150), His(151), Val(152) of thefollowing sequences of human IL-17F and the corresponding sequence foundin human IL-17A, as shown below. These residues are predicted to be onthe surface of the bioactive cytokine and therefore to be accessible tothe binding of an antibody of the present invention or equivalentprotein binding antagonist.

(Thr146-Val152 of SEQ ID NO: 4) hIL-17F TPVIHHV(Thr148-Val154 of SEQ ID NO: 2) hIL-17A TPIVHHV

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope is a discontinuous epitopecomprising residues from two separate peptide chains of human IL-17F, asshown below; or the equivalent sequence found in human IL-17A, as shownbelow. Specifically, residues 105-109, 147-152 of hIL-17F and 107-111,148-154 of hIL-17A are predicted to be on the surface of the bioactivecytokine and therefore are accessible to the binding of an antibody ofthe present invention or equivalent protein binding antagonist.

hIL-17F Sequences (Asp105-Asn109 [DISMN] and Pro147-Val152 [PVIHHV]of SEQ ID NO: 4) hIL-17A Sequences (Asp107-Asn111 [DYHMN]and Pro149-Val154 [PIVHHV] of SEQ ID NO: 2)

Optionally, the IL-17A/F antibodies bind to another epitope on bothIL-17A and IL-17F, wherein said epitope is a discontinuous epitopecomprising residues of two or three separate peptide chains of humanIL-17F, as shown below; or the equivalent sequence found in humanIL-17A. Specifically, residues 81, 82, 121, 132, 134 of hIL-17F and 83,84, 123, 134, 136 of hIL-17A are predicted to be on the surface of thebioactive cytokine and therefore to be accessible to the binding of anantibody of the present invention or equivalent protein bindingantagonist.

hIL-17F Sequences (Asp79-Ser85 [DPNRYPS] and Val119-Arg122 [VVRR] andSer130-Glu134 [SFQLE] of SEQ ID NO: 4)hIL-17A Sequences (Asp81-Ser87 [DPERYPS] and Val121-Arg124 [VLRR] andSer132-Glu136 [SFRLE] of SEQ ID NO: 2)

In a particular embodiment, the present invention provides bispecificantibodies that bind both IL-17A and IL-17F. Bispecific antibodies(BsAbs) are antibodies that have two different antigen binding sites,such that the antibody specifically binds to two different antigens.Antibodies having higher valencies (i.e., the ability to bind to morethan two antigens) can also be prepared; they are referred to asmultispecific antibodies.

The bispecific antibody preferably is a monoclonal antibody (MAb). Inparticular embodiments, the antibody is chimeric, or humanized, or fullyhuman. Fully human antibodies may be generated by procedures thatinvolve immunizing transgenic mice, wherein human immunoglobulin geneshave been introduced into the mice, as discussed below. Bispecificantibodies of the invention, which bind IL-17A and IL-17F, are referredto herein as bispecific IL-17A/F antibodies or bispecific A/F MAbs.

In yet other particular embodiments, there is provided the hybridomacell line which produces monoclonal antibodies of the present invention.In another embodiment, the IL-17A/F antibodies are linked to one or morenon-proteinaceous polymers selected from the group consisting ofpolyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to acytotoxic agent or enzyme, or to a radioisotope, fluorescent compound orchemiluminescent compound.

Typical methods of the invention include methods to treat pathologicalconditions or diseases in mammals associated with or resulting fromincreased or enhanced IL-17A or IL-17F expression and/or activity. Inthe methods of treatment, IL-17A/F antibodies may be administered whichpreferably block or reduce the respective receptor binding or activationto their receptor(s). Optionally, the IL-17A/F antibodies employed inthe methods will be capable of blocking or neutralizing the activity ofboth IL-17A and IL-17F, e.g., a dual antagonist which blocks orneutralizes activity of both IL-17A or IL-17F (i.e. a cross-reactiveIL-17A/F antibody as described herein). The methods contemplate the useof a single, cross-reactive antibody or a combination of two or moreantibodies.

The invention also provides compositions which comprise IL-17A/Fantibodies. Optionally, the compositions of the invention will includepharmaceutically acceptable carriers or diluents. Preferably, thecompositions will include one or more Il-17A/F antibodies in an amountwhich is therapeutically effective to treat a pathological condition ordisease.

As such, the present invention concerns compositions and methods usefulfor the diagnosis and treatment of immune related disease in mammals,including humans. The present invention is based on the identificationof antibodies that bind to both IL-17A and IL-17F (including agonist andantagonist antibodies) which either stimulate or inhibit the immuneresponse in mammals. Immune related diseases can be treated bysuppressing or enhancing the immune response. Antibodies that enhancethe immune response stimulate or potentiate the immune response to anantigen. Antibodies which stimulate the immune response can be usedtherapeutically where enhancement of the immune response would bebeneficial. Alternatively, antibodies that suppress the immune responseattenuate or reduce the immune response to an antigen (e.g.,neutralizing antibodies) can be used therapeutically where attenuationof the immune response would be beneficial (e.g., inflammation).

Accordingly, antibodies that bind both IL-17A and IL-17F (also referredto herein as IL-17A/F, A/F and/or cross-reactive IL-17A and IL-17Fantibodies) of the present invention and are also useful to preparemedicines and medicaments for the treatment of immune-related andinflammatory diseases, including for example, systemic lupuserythematosis, arthritis, psoriatic arthritis, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis, idiopathic inflammatory myopathies, Sjogren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, thyroiditis, diabetes mellitus,immune-mediated renal disease, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious, autoimmune chronic active hepatitis, primary biliarycirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease, colitis, Crohn's disease gluten-sensitiveenteropathy, and endotoxemia, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme and atopicand contact dermatitis, psoriasis, neutrophilic dermatoses, cysticfibrosis, allergic diseases such as asthma, allergic rhinitis, foodhypersensitivity and urticaria, cystic fibrosis, immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis,adult respiratory disease (ARD), acute respiratory distress syndrome(ARDS) and inflammatory lung injury such as asthma, chronic obstructivepulmonary disease (COPD), airway hyper-responsiveness, chronicbronchitis, allergic asthma and hypersensitivity pneumonitis,transplantation associated diseases including graft and organ rejectionand graft-versus-host-disease, septic shock, multiple organ failure,cancer and angiogenesis.

In a specific aspect, such medicines and medicaments comprise atherapeutically effective amount of an IL-17A/F antibody with apharmaceutically acceptable carrier. Preferably, the admixture issterile.

In one aspect, the present invention concerns an isolated antibody whichbinds to both IL-17A and IL-17F. In another aspect, the antibody mimicsthe activity of both IL-17A and IL-17F (an agonist antibody) orconversely the antibody inhibits or neutralizes the activity of bothIL-17A and IL-17F (an antagonist antibody). In another aspect, theantibody is a monoclonal antibody, which preferably has nonhumancomplementarity determining region (CDR) residues and human frameworkregion (FR) residues.

In a further embodiment, the invention concerns a method of identifyingagonist or antagonist antibodies of Il-17A and IL-17F, said methodcomprising contacting both IL-17A and IL-17F with a candidate moleculeand monitoring a biological activity mediated by IL-17A and/or IL-17F.In another embodiment, the invention concerns a composition of mattercomprising an IL-17A/F agonist or antagonist antibody which binds bothIL-17A and IL-17F in admixture with a carrier or excipient. In oneaspect, the composition comprises a therapeutically effective amount ofthe IL-17A/F antibody. In another aspect, when the composition comprisessuch an agonistic IL-17A/F antibody, the composition is useful for: (a)enhancing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) stimulating or enhancing an immune response in amammal in need thereof, (c) increasing the proliferation ofT-lymphocytes in a mammal in need thereof in response to an antigen, (d)stimulating the activity of T-lymphocytes or (e) increasing the vascul &permeability. In a further aspect, when the composition comprises suchan antagonistic IL-17A/F antibody, the composition is useful for: (a)decreasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) inhibiting or reducing an immune response in amammal in need thereof, (c) decreasing the activity of T-lymphocytes or(d) decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In another aspect, the compositioncomprises a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

In another embodiment, the invention concerns a method of treating animmune related disorder in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of anagonistic or antagonistic IL-17A/F antibody.

In a preferred aspect, the immune related disorder is selected form thegroup consisting of systemic lupus erythematosis, arthritis, psoriaticarthritis, rheumatoid arthritis, osteoarthritis, juvenile chronicarthritis, spondyloarthropathies, systemic sclerosis, idiopathicinflammatory myopathies, Sjogren's syndrome, systemic vasculitis,sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia,thyroiditis, diabetes mellitus, immune-mediated renal disease,demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barre syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious, autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease,colitis, Crohn's disease gluten-sensitive enteropathy, and endotoxemia,autoimmune or immune-mediated skin diseases including bullous skindiseases, erythema multiforme and atopic and contact dermatitis,psoriasis, neutrophilic dermatoses, cystic fibrosis, allergic diseasessuch as asthma, allergic rhinitis, food hypersensitivity and urticaria,cystic fibrosis, immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis, adult respiratory disease(ARD), acute respiratory distress syndrome (ARDS) and inflammatory lunginjury such as asthma, chronic obstructive pulmonary disease (COPD),airway hyper-responsiveness, chronic bronchitis, allergic asthma andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft and organ rejection and graft-versus-host-disease,septic shock, multiple organ failure, cancer and angiogenesis.

Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody. In another embodiment, theinvention provides an antibody which specifically binds to both IL-17Aand IL-17F. The antibody may be labeled and may be immobilized on asolid support. In a further aspect, the antibody is an antibodyfragment, a monoclonal antibody, a single-chain antibody, or ananti-idiotypic antibody.

In still another embodiment, the invention concerns an isolatedpolynucleotide that encodes a polypeptide of the present invention,wherein said polypeptide is capable of binding to both IL-17A andIL-17F.

In still another embodiment, the invention concerns an isolatedpolypeptide of the present invention, wherein said polypeptide iscapable of binding to both IL-17A and IL-17F.

Processes for producing the same are also herein described, whereinthose processes comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of said antibody and recovering saidantibody from the cell culture.

In yet another embodiment, the present invention provides a compositioncomprising an anti-IL-17A/F antibody in admixture with apharmaceutically acceptable carrier. In one aspect, the compositioncomprises a therapeutically effective amount of the antibody.Preferably, the composition is sterile. The composition may beadministered in the form of a liquid pharmaceutical formulation, whichmay be preserved to achieve extended storage stability. Alternatively,the antibody is a monoclonal antibody, an antibody fragment, a humanizedantibody, or a single-chain antibody.

In a further embodiment, the invention concerns an article ofmanufacture, comprising: (a) a composition of matter comprising anIL-17A/F antibody, or an antibody that specifically binds to both IL-17Aand IL-17F; (b) a container containing said composition; and (c) a labelaffixed to said container, or a package insert included in saidcontainer referring to the use of said IL-17A/F antibody thereof in thetreatment of an immune related disease. The composition may comprise atherapeutically effective amount of the IL-17A/F antibody.

In yet another embodiment, the present invention concerns a method ofdiagnosing an immune related disease in a mammal, comprising detectingthe level of expression of a gene encoding either or both IL-17A and/orIL-17F (a) in a test sample of tissue cells obtained from the mammal,and (b) in a control sample of known normal tissue cells of the samecell type, wherein a higher or lower expression level in the test sampleas compared to the control sample indicates the presence of immunerelated disease in the mammal from which the test tissue cells wereobtained.

In another embodiment, the present invention concerns a method ofdiagnosing an immune disease in a mammal, comprising (a) contacting anIL-17A/F antibody with a test sample of tissue cells obtained from themammal, and (b) detecting the formation of a complex between theantibody and either or both IL-17A and IL-17F in the test sample;wherein the formation of said complex is indicative of the presence orabsence of said disease. The detection may be qualitative orquantitative, and may be performed in comparison with monitoring thecomplex formation in a control sample of known normal tissue cells ofthe same cell type. A larger quantity of complexes formed in the testsample indicates the presence or absence of an immune disease in themammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. The test sample isusually obtained from an individual suspected of having a deficiency orabnormality of the immune system.

In another embodiment, the invention provides a method of diagnosing animmune-related disease in a mammal which comprises detecting thepresence or absence of both IL-17A and IL-17F in a test sample of tissuecells obtained from said mammal, wherein the presence or absence of bothIL-17A and IL-17F in said test sample is indicative of the presence ofan immune-related disease in said mammal.

In a still further embodiment, the invention provides a method forenhancing the infiltration of inflammatory cells from the vasculatureinto a tissue of a mammal comprising administering to said mammal (a) anIL-17A/F agonist antibody, wherein the infiltration of inflammatorycells from the vasculature in the mammal is enhanced. In a still furtherembodiment, the invention provides a method for decreasing theinfiltration of inflammatory cells from the vasculature into a tissue ofa mammal comprising administering to said mammal an antagonist IL-17A/Fantibody, wherein the infiltration of inflammatory cells from thevasculature in the mammal is decreased.

In a still further embodiment, the invention provides a method ofincreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal an IL-17A/F agonist antibody, wherein theactivity of T-lymphocytes in the mammal is increased.

In a still further embodiment, the invention provides a method ofdecreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal an IL-17A/F antagonist antibody, whereinthe activity of T-lymphocytes in the mammal is decreased.

In a still further embodiment, the invention provides a method ofincreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal an IL-17A/F agonist antibody, wherein theproliferation of T-lymphocytes in the mammal is increased.

In a still further embodiment, the invention provides a method ofdecreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) an IL-17A/F antagonist antibody,wherein the proliferation of T-lymphocytes in the mammal is decreased.

The invention also provides articles of manufacture and kits whichinclude one or more IL-17A/F antibodies.

B) DEFINITIONS

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.Thus, as used herein, the term “antibody” or “antibody peptide(s)”refers to an intact antibody, or a binding fragment thereof thatcompetes with the intact antibody for specific binding and includeschimeric, humanized, fully human, and bispecific antibodies. In certainembodiments, binding fragments are produced by recombinant DNAtechniques. In additional embodiments, binding fragments are produced byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, andsingle-chain antibodies. “Native antibodies and immunoglobulins” areusually heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light (L) chains and two identical heavy (H)chains. Each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide-linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (VH) followed by a numberof constant domains. Each light chain has a variable domain at one end(VL) and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Particular amino acid residues are believedto form an interface between the light- and heavy-chain variable domains(Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc.Natl. Acad. Sci. U.S.A. 82:4592 (1985)).

The term “isolated antibody” as used herein refers to an antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

A “variant” anti-IL-17A and/or IL-17F and/or IL-17A/F antibody, refersherein to a molecule which differs in amino acid sequence from a“parent” anti-IL-17A and/or IL-17F and/or IL-17A/F antibody amino acidsequence by virtue of addition, deletion and/or substitution of one ormore amino acid residue(s) in the parent antibody sequence. In thepreferred embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more hypervariable region(s) of the parentantibody. For example, the variant may comprise at least one, e.g. fromabout one to about ten, and preferably from about two to about five,substitutions in one or more hypervariable regions of the parentantibody. Ordinarily, the variant will have an amino acid sequencehaving at least 75% amino acid sequence identity with the parentantibody heavy or light chain variable domain sequences, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, and most preferably at least 95%. Identity or homology with respectto this sequence is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the parentantibody residues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence shall be construed as affecting sequenceidentity or homology. The variant retains the ability to bind humanIL-17A and/or IL-17F and preferably has properties which are superior tothose of the parent antibody. For example, the variant may have astronger binding affinity, enhanced ability to inhibit IL-17A and/orIL-17F-induced inflammation. To analyze such properties, one shouldcompare a Fab form of the variant to a Fab form of the parent antibodyor a full length form of the variant to a full length form of the parentantibody, for example, since it has been found that the format of theanti-IL-17A and/or IL-17F and/or IL-17A/F antibody impacts its activityin the biological activity assays disclosed herein. The variant antibodyof particular interest herein is one which displays at least about 10fold, preferably at least about 20 fold, and most preferably at leastabout 50 fold, enhancement in biological activity when compared to theparent antibody.

The term “parent antibody” as used herein refers to an antibody which isencoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or human antibody.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule.

The term “bind(ing) of a polypeptide of the invention to a ligand”includes, but is not limited to, the binding of a ligand polypeptide ofthe present invention to a receptor; the binding of a receptorpolypeptide of the present invention to a ligand; the binding of anantibody of the present invention to an antigen or epitope; the bindingof an antigen or epitope of the present invention to an antibody; thebinding of an antibody of the present invention to an anti-idiotypicantibody; the binding of an anti-idiotypic antibody of the presentinvention to a ligand; the biding of an anti-idiotypic antibody of thepresent invention to a receptor; the binding of an anti-anti-idiotypicantibody of the present invention to a ligand, receptor or antibody,etc.

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol.79:315-321; Kostelny et al. (1992), J. Immunol. 148:1547-1553.

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used. Specifically, a chimeric antibody is produced byrecombinant DNA technology in which all or part of the hinge andconstant regions of an immunoglobulin light chain, heavy chain, or both,have been substituted for the corresponding regions from anotheranimal's immunoglobulin light chain or heavy chain. In this way, theantigen-binding portion of the parent monoclonal antibody is graftedonto the backbone of another species antibody. One approach, describedin EP 0239400 to Winter et al. describes the substitution of onespecies' complementarity determining regions (CDRs) for those of anotherspecies, such as substituting the CDRs from human heavy and light chainimmunoglobulin variable region domains with CDRs from mouse variableregion domains. These altered antibodies may subsequently be combinedwith human immunoglobulin constant regions to form antibodies that arehuman except for the substituted murine CDRs which are specific for theantigen. Methods for grafting CDR regions of antibodies may be found,for example in Riechmann et al. (1988) Nature 332:323-327 and Verhoeyenet al. (1988) Science 239:1534-1536.

The term “effective neutralizing titer” as used herein refers to theamount of antibody which corresponds to the amount present in the serumof animals (human or cotton rat) that has been shown to be eitherclinically efficacious (in humans) or to reduce virus by 99% in, forexample, cotton rats. The 99% reduction is defined by a specificchallenge of, e.g., 10³ pfu, 10⁴ pfu, 10⁵ pfu, 10⁶ pfu, 10⁷ pfu, 10⁸pfu, or 10⁹ pfu) of RSV.

As used herein, the term “epitope” refers to the portion of an antigento which a monoclonal antibody specifically binds. Thus, the term“epitope” includes any protein determinant capable of specific bindingto an immunoglobulin or T-cell receptor. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. More specifically, the term “IL-17A epitope,” “IL-17Fepitope” and/or “IL-17A/F epitope” as used herein refers to a portion ofthe corresponding polypeptide having antigenic or immunogenic activityin an animal, preferably in a mammal, and most preferably in a mouse ora human. An epitope having immunogenic activity is a portion of anIL-17A and/or IL-17F polypeptide that elicits an antibody response in ananimal. An epitope having antigenic activity is a portion of an IL-17Aand/or IL-17F polypeptide to which an antibody immunospecifically bindsas determined by any method well known in the art, for example, byimmunoassays. Antigenic epitopes need not necessarily be immunogenic.Such epitopes can be linear in nature or can be a discontinuous epitope.Thus, as used herein, the term “conformational epitope” refers to adiscontinuous epitope formed by a spatial relationship between aminoacids of an antigen other than an unbroken series of amino acids. Morespecifically, the term epitope encompasses the epitopes as definedherein, as they apply to both IL-17A and IL-17F.

The term “epitope tagged” when used herein refers to the anti-IL-17Aand/or IL-17F and/or IL-17A/F antibody fused to an “epitope tag”. Theepitope tag polypeptide has enough residues to provide an epitopeagainst which an antibody can be made, yet is short enough such that itdoes not interfere with activity of antibodies of the present invention.The epitope tag preferably is sufficiently unique so that the antibodythereagainst does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least 6 amino acid residuesand usually between about 8-50 amino acid residues (preferably betweenabout 9-30 residues). Examples include the flu HA tag polypeptide andits antibody 12CA5 (Field et al. Mol. Cell. Biol. 8:2159-2165 (1988));the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto(Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,Protein Engineering 3(6):547-553 (1990)). In certain embodiments, theepitope tag is a “salvage receptor binding epitope”. As used herein, theterm “salvage receptor binding epitope” refers to an epitope of the Fcregion of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

The term “fragment” as used herein refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of aIl-17A or IL-17F polypeptide or an antibody that immunospecificallybinds to a either Il-17A or IL-17F or both IL-17A and IL-17Fpolypeptide.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH2-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes (about 330 amino acids). Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM,IgA, IgD and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. (See generally, Fundamental Immunology (Paul,W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated byreference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk, 1987, J. Mol. Biol. 196: 901-917) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes and antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and(Fab′)₂, as well as diabodies, linear antibodies, multivalent ormultispecific hybrid antibodies (as described above and in detail in:Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in singlechains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), whichare incorporated herein by reference). (See, generally, Hood et al.,“Immunology,” Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,Nature, 323, 15-16 (1986), which are incorporated herein by reference).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding. Single chain antibodies are discussedin detail by Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994). Various methods of generating single chain antibodiesare known, including those described in U.S. Pat. Nos. 4,694,778 and5,260,203; International Patent Application Publication No. WO 88/01649;Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra etal. (1988) Science 242:1038-1041, the disclosures of which areincorporated by reference for any purpose. In specific embodiments,single-chain antibodies can also be bi-specific and/or humanized.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least thevariable domains of the immunoglobulin heavy and light chains. Animmunologically functional immunoglobulin fragment of the invention iscapable of binding to a ligand, preventing binding of the ligand to itsreceptor, interrupting the biological response resulting from ligandbinding to the receptor, or any combination thereof. Preferably, animmunologically functional immunoglobulin fragment of the inventionbinds specifically to both IL-17A and IL-17F.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB3; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that producesIL-17RA from an expression vector. In contrast, IL-17RA can be producedby a cell that is a “natural source” of IL-17RA, and that lacks anexpression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a IL-17RApolypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of IL-17RAusing affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel. However,the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and the like, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for IL-17A or IL-17F” is anoligonucleotide having a sequence (a) capable of forming a stabletriplex with a portion of the IL-17A or IL-17F gene, or (b) capable offorming a stable duplex with a portion of an mRNA transcript of theIL-17A or IL-17F gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

C) ANTIBODIES THAT BIND IL-17A AND IL-17F

The antibodies of the invention specifically bind to both IL-17A andIL17F. In some embodiments, the antibodies of the invention specificallybind a monomeric form of both IL-17A and IL-17F. In some embodiments,the antibodies of the invention bind a homodimeric form of either IL-17Aor IL-17F. In still other embodiments, the antibodies of the inventionspecifically bind a multimeric form of IL-17A and IL-17F (e.g., aheterodimeric form). Preferred antibodies of the invention block abiological activity of both IL-17A and IL-17F.

Preferred antibodies, and antibodies suitable for use in the method ofthe invention, include, for example, fully human antibodies, humanantibody homologs, humanized antibody homologs, chimeric antibodyhomologs, Fab, Fab′, F(ab′)₂ and F(v) antibody fragments, single chainantibodies, and monomers or dimers of antibody heavy or light chains ormixtures thereof. Antibodies of the invention are preferably monoclonalantibodies.

The antibodies of the invention may include intact immunoglobulins ofany isotype including types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof). The antibodies preferably include intact IgG and morepreferably IgG1. The light chains of the immunoglobulin may be kappa orlambda. The light chains are preferably kappa.

The antibodies of the invention include portions of intact antibodiesthat retain antigen-binding specificity, for example, Fab fragments,Fab′ fragments, F(ab′)₂ fragments, F(v) fragments, heavy chain monomersor dimers, light chain monomers or dimers, dimers consisting of oneheavy and one light chain, and the like. Thus, antigen bindingfragments, as well as full-length dimeric or trimeric polypeptidesderived from the above-described antibodies are themselves useful.

The direct use of rodent monoclonal antibodies (MAbs) as humantherapeutic agents led to human anti-rodent antibody (“HARA”) (forexample, human anti-mouse antibody (“HAMA”)) responses which occurred ina significant number of patients treated with the rodent-derivedantibody (Khazaeli, et al., (1994) Immunother. 15:42-52). Chimericantibodies containing fewer murine amino acid sequences are believed tocircumvent the problem of eliciting an immune response in humans.

Refinement of antibodies to avoid the problem of HARA responses led tothe development of “humanized antibodies.” Humanized antibodies areproduced by recombinant DNA technology, in which at least one of theamino acids of a human immunoglobulin light or heavy chain that is notrequired for antigen binding has been substituted for the correspondingamino acid from a nonhuman mammalian immunoglobulin light or heavychain. For example, if the immunoglobulin is a mouse monoclonalantibody, at least one amino acid that is not required for antigenbinding is substituted using the amino acid that is present on acorresponding human antibody in that position. Without wishing to bebound by any particular theory of operation, it is believed that the“humanization” of the monoclonal antibody inhibits human immunologicalreactivity against the foreign immunoglobulin molecule.

As a non-limiting example, a method of performing complementaritydetermining region (CDR) grafting may be performed by sequencing themouse heavy and light chains of the antibody of interest that binds tothe target antigen (e.g., IL-17A and IL-17F) and genetically engineeringthe CDR DNA sequences and imposing these amino acid sequences tocorresponding human V regions by site directed mutagenesis. Humanconstant region gene segments of the desired isotype are added, and the“humanized” heavy and light chain genes are co-expressed in mammaliancells to produce soluble humanized antibody. A typical expression cellis a Chinese Hamster Ovary (CHO) cell. Suitable methods for creating thechimeric antibodies may be found, for example, in Jones et al. (1986)Nature 321:522-525; Riechmann (1988) Nature 332:323-327; Queen et al.(1989) Proc. Nat. Acad. Sci. USA 86:10029; and Orlandi et al. (1989)Proc. Natl. Acad. Sci. USA 86:3833.

Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033 and WO90/07861 describe the preparation of a humanized antibody. Human andmouse variable framework regions were chosen for optimal proteinsequence homology. The tertiary structure of the murine variable regionwas computer-modeled and superimposed on the homologous human frameworkto show optimal interaction of amino acid residues with the mouse CDRs.This led to the development of antibodies with improved binding affinityfor antigen (which is typically decreased upon making CDR-graftedchimeric antibodies). Alternative approaches to making humanizedantibodies are known in the art and are described, for example, inTempest (1991) Biotechnology 9:266-271.

The antibodies of the invention may be used alone or as immunoconjugateswith a cytotoxic agent. In some embodiments, the agent is achemotherapeutic agent. In some embodiments, the agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, modified Pseudomonas enterotoxin A, calicheamicin,adriamycin, 5-fluorouracil, and the like. Methods of conjugation ofantibodies and antibody fragments to such agents are known in theliterature.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto its epitope. Examples of suitable derivatives include, but are notlimited to fucosylated antibodies and fragments, glycosylated antibodiesand fragments, acetylated antibodies and fragments, pegylated antibodiesand fragments, phosphorylated antibodies and fragments, and amidatedantibodies and fragments. The antibodies and derivatives thereof of theinvention may themselves by derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., block the binding of IL-17Aand/or IL-17F to their respective receptors, block the biologicalactivity of IL-17A and IL-17F, binding affinity) of the antibodies ofthe invention. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include, inter alia: (a) variants in which one ormore amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art. Antibodies of the invention also include antibody fragments.A “fragment” refers to polypeptide sequences which are preferably atleast about 40, more preferably at least to about 50, more preferably atleast about 60, more preferably at least about 70, more preferably atleast about 80, more preferably at least about 90, and more preferablyat least about 100 amino acids in length, and which retain somebiological activity or immunological activity of the full-lengthsequence, for example, the ability to block the binding of IL-17A and/orIL-17F to their respective receptors, block the biological activity ofIL-17A and IL-17F, binding affinity.

The invention also encompasses fully human antibodies such as thosederived from peripheral blood mononuclear cells of ovarian, breast,renal, colorectal, lung, endometrial, or brain cancer patients. Suchcells may be fused with myeloma cells, for example, to form hybridomacells producing fully human antibodies against both IL-17A and IL-17F.

The invention also encompasses bispecific antibodies that bind to bothIL-17A and IL-17F.

The antibodies of the invention are preferably nontoxic as demonstrated,for example, in in vivo toxicology studies.

The antibodies and derivatives thereof of the invention have bindingaffinities that include a dissociation constant (K_(d)) of less than1×10⁻². In some embodiments, the K_(d) is less than 1×10⁻³. In otherembodiments, the K_(d) is less than 1×10⁻⁴. In some embodiments, theK_(d) is less than 1×10⁻⁵. In still other embodiments, the K_(d) is lessthan 1×10⁻⁶. In other embodiments, the K_(d) is less than 1×10⁻⁷. Inother embodiments, the K_(d) is less than 1×10⁻⁸. In other embodiments,the K_(d) is less than 1×10⁻⁹. In other embodiments, the K_(d) is lessthan 1×10⁻¹⁰. In still other embodiments, the K_(d) is less than1×10⁻¹³. In some embodiments, the K_(d) is less than 1×10⁻¹². In otherembodiments, the K_(d) is less than 1×10⁻¹³. In other embodiments, theK_(d) is less than 1×10⁻¹⁴. In still other embodiments, the K_(d) isless than 1×10⁻¹⁵.

D) NUCLEIC ACIDS

The invention also includes nucleic acids encoding the heavy chainand/or light chain of the antibodies of the invention. Nucleic acids ofthe invention include nucleic acids having at least 80%, more preferablyat least about 90%, more preferably at least about 95%, and mostpreferably at least about 98% homology to nucleic acids of theinvention. The terms “percent similarity,” “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program. Nucleicacids of the invention also include complementary nucleic acids. In someinstances, the sequences will be fully complementary (no mismatches)when aligned. In other instances, there may be up to about a 20%mismatch in the sequences. In some embodiments of the invention areprovided nucleic acids encoding both a heavy chain and a light chain ofan antibody of the invention.

Nucleic acids of the invention can be cloned into a vector, such as aplasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) orvirus, into which another genetic sequence or element (either DNA orRNA) may be inserted so as to bring about the replication of theattached sequence or element. In some embodiments, the expression vectorcontains a constitutively active promoter segment (such as but notlimited to CMV, SV40, Elongation Factor or LTR sequences) or aninducible promoter sequence such as the steroid inducible pIND vector(Invitrogen), where the expression of the nucleic acid can be regulated.Expression vectors of the invention may further comprise regulatorysequences, for example, an internal ribosomal entry site. The expressionvector can be introduced into a cell by transfection, for example.

E) METHODS OF PRODUCING ANTIBODIES TO IL-17A AND IL-17F

The invention also provides methods of producing monoclonal antibodiesthat specifically bind to both IL-17A and IL-17F. Antibodies of theinvention may be produced in vivo or in vitro. One strategy forgenerating antibodies against both IL-17A and IL-17F involves immunizinganimals with both IL-17A and IL-17F. In some embodiments, animals areimmunized with the monomeric or multimeric form of FR both IL-17A andIL-17F. Animals so immunized will produce antibodies against both IL-17Aand IL-17F, as well as cross-reactive antibodies against both IL-17A andIL-17F. Standard methods are known for creating monoclonal antibodiesincluding, but are not limited to, the hybridoma technique (see Kohler &Milstein, (1975) Nature 256:495-497); the trioma technique; the humanB-cell hybridoma technique (see Kozbor et al. (1983) Immunol. Today4:72) and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole, et al. in MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).

Both IL-17A and IL-17F may be purified from cells or from recombinantsystems using a variety of well-known techniques for isolating andpurifying proteins. For example, but not by way of limitation, bothIL-17A and IL-17F may be isolated based on the apparent molecular weightof the protein by running the protein on an SDS-PAGE gel and blottingthe proteins onto a membrane. Thereafter, the appropriate size bandcorresponding to either protein may be cut from the membrane and used asan immunogen in animals directly, or by first extracting or eluting theprotein from the membrane. As an alternative example, the protein may beisolated by size-exclusion chromatography alone or in combination withother means of isolation and purification.

The invention also provides methods of producing monoclonal antibodiesthat specifically bind to homodimeric, heterodimeric, and/or multimericforms of both IL-17A and IL-17F. These different forms may be purifiedfrom cells or from recombinant systems using a variety of well-knowntechniques for isolating and purifying proteins. For example, but not byway of limitation, both IL-17A and IL-17F may be isolated based on theapparent molecular weight of the protein by running the protein on anSDS-PAGE gel and blotting the proteins onto a membrane. Thereafter, theappropriate size band corresponding to each may be cut from the membraneand used as an immunogen in animals directly, or by first extracting oreluting the protein from the membrane. As an alternative example, theprotein may be isolated by size-exclusion chromatography alone or incombination with other means of isolation and purification.

Other means of purification are available in such standard referencetexts as Zola, Monoclonal Antibodies: Preparation And Use Of MonoclonalAntibodies And Engineered Antibody Derivatives (Basics: From BackgroundTo Bench) Springer-Verlag Ltd., New York, 2000; Basic Methods InAntibody Production And Characterization, Chapter 11, “AntibodyPurification Methods,” Howard and Bethell, Eds., CRC Press, 2000;Antibody Engineering (Springer Lab Manual.), Kontermann and Dubel, Eds.,Springer-Verlag, 2001.

For in vivo antibody production, animals are generally immunized witheither IL-17A or IL-17F or an immunogenic portion of either (e.g. sharedepitopes as described above). The antigen is generally combined with anadjuvant to promote immunogenicity. Adjuvants vary according to thespecies used for immunization. Examples of adjuvants include, but arenot limited to: Freund's complete adjuvant (“FCA”), Freund's incompleteadjuvant (“FIA”), mineral gels (e.g., aluminum hydroxide), surfaceactive substances (e.g., lysolecithin, pluronic polyols, polyanions),peptides, oil emulsions, keyhole limpet hemocyanin (“KLH”),dinitrophenol (“DNP”), and potentially useful human adjuvants such asBacille Calmette-Guerin (“BCG”) and corynebacterium parvum. Suchadjuvants are also well known in the art. Immunization may beaccomplished using well-known procedures. The dose and immunizationregimen will depend on the species of mammal immunized, its immunestatus, body weight, and/or calculated surface area, etc. Typically,blood serum is sampled from the immunized mammals and assayed foranti-IL-17A and IL-17F antibodies using appropriate screening assays asdescribed below, for example.

A common method for producing humanized antibodies is to graft CDRsequences from a MAb (produced by immunizing a rodent host) onto a humanIg backbone, and transfection of the chimeric genes into Chinese HamsterOvary (CHO) cells which in turn produce a functional Ab that is secretedby the CHO cells (Shields, R. L., et al. (1995) Anti-IgE monoclonalantibodies that inhibit allergen-specific histamine release. Int Arch.Allergy Immunol. 107:412-413). The methods described within thisapplication are also useful for generating genetic alterations within Iggenes or chimeric Igs transfected within host cells such as rodent celllines, plants, yeast and prokaryotes (Frigerio L, et al. (2000)Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin inplants. Plant Physiol. 123:1483-1494).

Splenocytes from immunized animals may be immortalized by fusing thesplenocytes (containing the antibody-producing B cells) with an immortalcell line such as a myeloma line. Typically, myeloma cell line is fromthe same species as the splenocyte donor. In one embodiment, theimmortal cell line is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). In someembodiments, the myeloma cells are negative for Epstein-Barr virus (EBV)infection. In preferred embodiments, the myeloma cells areHAT-sensitive, EBV negative and Ig expression negative. Any suitablemyeloma may be used. Murine hybridomas may be generated using mousemyeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O—Ag14 myeloma lines). These murine myeloma lines are available fromthe ATCC. These myeloma cells are fused to the donor splenocytespolyethylene glycol (“PEG”), preferably 1500 molecular weightpolyethylene glycol (“PEG 1500”). Hybridoma cells resulting from thefusion are selected in HAT medium which kills unfused and unproductivelyfused myeloma cells. Unfused splenocytes die over a short period of timein culture. In some embodiments, the myeloma cells do not expressimmunoglobulin genes.

Hybridomas producing a desired antibody which are detected by screeningassays such as those described below may be used to produce antibodiesin culture or in animals. For example, the hybridoma cells may becultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. These techniques and culture media are well known bythose skilled in the art. Alternatively, the hybridoma cells may beinjected into the peritoneum of an unimmunized animal. The cellsproliferate in the peritoneal cavity and secrete the antibody, whichaccumulates as ascites fluid. The ascites fluid may be withdrawn fromthe peritoneal cavity with a syringe as a rich source of the monoclonalantibody.

Another non-limiting method for producing human antibodies is describedin U.S. Pat. No. 5,789,650 which describes transgenic mammals thatproduce antibodies of another species (e.g., humans) with their ownendogenous immunoglobulin genes being inactivated. The genes for theheterologous antibodies are encoded by human immunoglobulin genes. Thetransgenes containing the unrearranged immunoglobulin encoding regionsare introduced into a non-human animal. The resulting transgenic animalsare capable of functionally rearranging the transgenic immunoglobulinsequences and producing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes. The B-cells from the transgenicanimals are subsequently immortalized by any of a variety of methods,including fusion with an immortalizing cell line (e.g., a myeloma cell).

The antibodies of the present invention may also be prepared in vitrousing a variety of techniques known in the art. For example, but not byway of limitation, fully human monoclonal antibodies against IL-17A andIL-17F may be prepared by using in vitro-primed human splenocytes(Boerner et al. (1991) J. Immunol. 147:86-95).

Alternatively, for example, the antibodies of the invention may beprepared by “repertoire cloning” (Persson et al. (1991) Proc. Nat. Acad.Sci. USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods141:227-236). Further, U.S. Pat. No. 5,798,230 describes preparation ofhuman monoclonal antibodies from human B antibody-producing B cells thatare immortalized by infection with an Epstein-Barr virus that expressesEpstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2, required forimmortalization, is then inactivated resulting in increased antibodytiters.

In another embodiment, antibodies of the invention are formed by invitro immunization of peripheral blood mononuclear cells (“PBMCs”). Thismay be accomplished by any means known in the art, such as, for example,using methods described in the literature (Zafiropoulos et al. (1997) J.Immunological Methods 200:181-190).

In a specific embodiment, bispecific and single chain antibodies thatbind both IL-17A and IL-17F are made. One method of the presentinvention is a method for producing a bispecific A/F antibody. Themethod comprises fusing hybridoma cells that secrete a monoclonalantibody that binds IL-17A, with hybridoma cells that secrete amonoclonal antibody that binds IL-17F, thereby preparing a hybridhybridoma that secretes a bispecific A/F monoclonal antibody. In oneembodiment, the method comprises fusing hybridoma cells that secrete anantagonistic (or agonistic) IL-17A MAb, with hybridoma cells thatsecrete an antagonistic (or agonistic) IL-17F MAb. Conventionaltechniques for conducting such a fusion, and for isolating the desiredhybrid hybridoma, include those described elsewhere herein, and thoseillustrated in the examples below.

U.S. Pat. No. 6,060,285 discloses a process for the production ofbispecific antibodies, in which at least the genes for the light chainand the variable portion of the heavy chain of an antibody having afirst specificity are transfected into a hybridoma cell secreting anantibody having a second specificity. When the transfected hybridomacells are cultured, bispecific antibodies are produced, and may beisolated by various means known in the art.

Other investigators have used chemical coupling of antibody fragments toprepare antigen-binding molecules having specificity for two differentantigens (Brennan et al., Science 229:81 1985; Glennie et al., J.Immunol. 139:2367, 1987). U.S. Pat. No. 6,010,902 also discussestechniques known in the art by which bispecific antibodies can beprepared, for example by the use of heterobifunctional cross-linkingreagents such as GMBS (maleimidobutryloxy succinimide) or SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate). (See, e.g., Hardy,“Purification And Coupling Of Fluorescent Proteins For Use In FlowCytometry,” Handbook Of Experimental Immunology, 4.sup.th Ed., Volume 1,Immunochemistry, Weir et al. (eds.), pp. 31.4-31.12, 1986).

The ability to produce antibodies via recombinant DNA technology hasfacilitated production of bispecific antibodies. Kostelny et al.utilized the leucine zipper moieties from the fos and jun proteins(which preferentially form heterodimers) to produce bispecificantibodies able to bind both the cell surface molecule CD3 and thereceptor for Interleukin-2 (J. Immunol. 148:1547; 1992).

Single chain antibodies may be formed by linking heavy and light chainvariable region (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable regionpolypeptides (V_(L) and V_(H)). The resulting antibody fragments canform dimers or higher oligomers, depending on such factors as the lengthof a flexible linker between the two variable domains (Kortt et al.,Protein Engineering 10:423, 1997). In particular embodiments, two ormore scFvs are joined by use of a chemical cross-linking agent.

Techniques developed for the production of single chain antibodies canbe adapted to produce single chain antibodies of the present invention,that bind both IL-17A and IL-17F. Such techniques include thosedescribed in U.S. Pat. No. 4,946,778; Bird (Science 242:423, 1988);Huston et al. (Proc. Natl. Acad. Sci. USA 85:5879, 1988); and Ward etal. (Nature 334:544, 1989). Once desired single chain antibodies areidentified (for example, from a phage-display library), those of skillin the art can further manipulate the DNA encoding the single chainantibody(ies) to yield bispecific antibodies, including bispecificantibodies having Fc regions.

Single chain antibodies against IL-17A and IL-17F may be concatamerizedin either order (i.e., anti-IL-17A-anti-IL-17F oranti-IL-17F-anti-IL-17A). In particular embodiments, starting materialsfor preparing a bispecific A/F antibody include an antagonistic (oragonistic) single chain antibody directed against IL-17A and anantagonistic (or agonistic) single chain antibody directed againstIL-17F.

U.S. Pat. No. 5,582,996 discloses the use of complementary interactivedomains (such as leucine zipper moieties or other lock and keyinteractive domain structures) to facilitate heterodimer formation inthe production of bispecific antibodies. The complementary interactivedomain(s) may be inserted between an Fab fragment and another portion ofa heavy chain (i.e., C_(H1) or C_(H2) regions of the heavy chain). Theuse of two different Fab fragments and complementary interactive domainsthat preferentially heterodimerize will result in bispecific antibodymolecules. Cysteine residues may be introduced into the complementaryinteractive domains to allow disulphide bonding between thecomplementary interactive domains and stabilize the resulting bispecificantibodies.

Tetravalent, bispecific molecules can be prepared by fusion of DNAencoding the heavy chain of an F(ab′)₂ fragment of an antibody witheither DNA encoding the heavy chain of a second F(ab′)₂ molecule (inwhich the CH1 domain is replaced by a CH3 domain), or with DNA encodinga single chain Fv fragment of an antibody, as described in U.S. Pat. No.5,959,083. Expression of the resultant fusion genes in mammalian cells,together with the genes for the corresponding light chains, yieldstetravalent bispecific molecules having specificity for selectedantigens.

Bispecific antibodies can also be produced as described in U.S. Pat. No.5,807,706, which is incorporated by reference herein. Generally, themethod involves introducing a protuberance in a first polypeptide and acorresponding cavity in a second polypeptide, polypeptides interface.The protuberance and cavity are positioned so as to promoteheteromultimer formation and hinder homomultimer formation. Theprotuberance is created by replacing amino acids having small sidechains with amino acids having larger side chains. The cavity is createdby the opposite approach, i.e., replacing amino acids having relativelylarge side chains with amino acids having smaller side chains.

The protuberance and cavity can be generated by conventional methods formaking amino acid substitutions in polypeptides. For example, a nucleicacid encoding a polypeptide may be altered by conventional in vitromutagenesis techniques. Alternatively, a polypeptide incorporating adesired amino acid substitution may be prepared by peptide synthesis.Amino acids chosen for substitution are located at the interface betweenthe first and second polypeptides.

F) SCREENING FOR ANTIBODY SPECIFICITY

Screening for antibodies that specifically bind to both IL-17A andIL-17F may be accomplished using an enzyme-linked immunosorbent assay(ELISA) in which microtiter plates are coated with both IL-17A andIL-17F. In some embodiments, antibodies that bind both IL-17A and IL-17Ffrom positively reacting clones can be further screened for reactivityin an ELISA-based assay using microtiter plates coated with the otherforms IL-17A and IL-17F, or other IL-17 family members. Clones thatproduce antibodies that are reactive to another forms or family membersare eliminated, and clones that produce antibodies that are reactive toboth IL-17A and IL-17F may be selected for further expansion anddevelopment. Confirmation of reactivity of the antibodies to both IL-17Aand IL-17F may be accomplished, for example, using a Western Blot assayin which protein from ovarian, breast, renal, colorectal, lung,endometrial, or brain cancer cells and purified FR-α and other folatereceptor isoforms are run on an SDS-PAGE gel, and subsequently areblotted onto a membrane. The membrane may then be probed with theputative anti-FR-α antibodies. Reactivity with both IL-17A and IL-17Fand not another family member confirms specificity of reactivity forboth IL-17A and IL-17F.

In some embodiments, the binding affinity of the antibodies of thepresent invention antibodies is determined. Antibodies of the inventionpreferably have a binding affinity to both IL-17A and IL-17F of at leastabout 1×10⁻⁷ M, more preferably at least about 1×10⁻⁸ M, more preferablyat least about 1×10⁻⁹ M, and most preferably at least about 1×10⁻⁸ M.Preferred antibody-producing cells of the invention producesubstantially only antibodies having a binding affinity to both IL-17Aand IL-17F of at least about 1×10⁻⁷ M, more preferably at least about1×10⁻⁸ M, more preferably at least about 1×10⁻⁹ M, and most preferablyat least about 1×10⁻¹⁰ M. Preferred compositions of the inventioncomprise substantially only antibodies having a binding affinity to bothIL-17A and IL-17F of at least about 1×10⁻⁷ M, more preferably at leastabout 1×10⁻⁸ M, more preferably at least about 1×10⁻⁹ M, and mostpreferably at least about 1×10⁻¹⁰ M.

The antibodies of the invention preferably induce antibody-dependentcellular cytotoxicity (ADCC) in IL-17RA and IL-17RC-bearing cells. ADCCassays are known in the art.

G) ANTI-IL-17A AND IL-17F ANTIBODY-PRODUCING CELLS

Antibody-producing cells of the invention include any insect expressioncell line known, such as for example, Spodoptera frugiperda cells. Theexpression cell lines may also be yeast cell lines, such as, forexample, Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.The expression cells may also be mammalian cells such as, for example,hybridoma cells (e.g., NS0 cells), Chinese hamster ovary cells, babyhamster kidney cells, human embryonic kidney line 293, normal dog kidneycell lines, normal cat kidney cell lines, monkey kidney cells, Africangreen monkey kidney cells, COS cells, and non-tumorigenic mouse myoblastG8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3cells, LMTK31 cells, mouse sertoli cells, human cervical carcinomacells, buffalo rat liver cells, human lung cells, human liver cells,mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells.

In some preferred embodiments, the antibody-producing cells of theinvention produce antibodies that specifically bind to both IL-17A andIL-17F. The cells preferably are substantially free of both IL-17A andIL-17F binding competitors. In preferred embodiments, theantibody-producing cells comprise less than about 10%, preferably lessthan about 5%, more preferably less than about 1%, more preferably lessthan about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight both IL-17A and IL-17F binding competitors. Insome preferred embodiments, the antibodies produced by theantibody-producing cells are substantially free of both IL-17A andIL-17F competitors. In preferred embodiments, antibodies produced by theantibody-producing cells comprise less than about 10%, preferably lessthan about 5%, more preferably less than about 1%, more preferably lessthan about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight both IL-17A and IL-17F binding competitors.Preferred antibody-producing cells of the invention producesubstantially only antibodies having a binding affinity to both IL-17Aand IL-17F of at least about 1×10⁻⁷ M, more preferably at least about1×10⁻⁸ M, more preferably at least about 1×10⁻⁹ M, and most preferablyat least about 1×10⁻¹⁰ M.

H) ANTIBODY PURIFICATION

Methods of antibody purification are known in the art. In someembodiments of the invention, methods for antibody purification includefiltration, affinity column chromatography, cation exchangechromatography, anion exchange chromatography, and concentration. Thefiltration step preferably comprises ultrafiltration, and morepreferably ultrafiltration and diafiltration. Filtration is preferablyperformed at least about 5-50 times, more preferably 10 to 30 times, andmost preferably 14 to 27 times. Affinity column chromatography, may beperformed using, for example, PROSEP Affinity Chromatography (Millipore,Billerica, Mass.). In a preferred embodiment, the affinitychromatography step comprises PROSEP-VA column chromatography. Eluatemay be washed in a solvent detergent. Cation exchange chromatography mayinclude, for example, SP-Sepharose Cation Exchange Chromatography. Anionexchange chromatography may include, for example but not limited to,Q-Sepharose Fast Flow Anion Exchange. The anion exchange step ispreferably non-binding, thereby allowing removal of contaminantsincluding DNA and BSA. The antibody product is preferably nanofiltered,for example, using a Pall DV 20 Nanofilter. The antibody product may beconcentrated, for example, using ultrafiltration and diafiltration. Themethod may further comprise a step of size exclusion chromatography toremove aggregates.

I) THERAPEUTIC USES OF THE CROSS-REACTIVE IL-17A AND IL-17F ANTIBODIES

Antibodies that cross-reactive to both IL-17A and IL-17F can be used tomodulate the immune system by binding IL-17A and IL-17F (either singlyor together), and thus, preventing the binding of IL-17A with eitherIL-17RA or IL-17RC and IL-17F with IL-17RC or any other receptor thatthey may bind, especially an IL-17 family member. The antibodies of theinvention can also be used to modulate the immune system by inhibitingthe binding of both IL-17A with the endogenous IL-17RA and/or IL-17RCreceptor and IL-17F with the endogenous IL-17RC receptor. The antibodiesof the invention can be also used to treat a subject which produces anexcess of either IL-17A and/or IL-17F. Suitable subjects includemammals, such as humans. For example, the antibodies of the inventionare useful in binding, blocking, inhibiting, reducing, antagonizing orneutralizing of both IL-17A and IL-17F (either singly or together), inthe treatment of inflammation and inflammatory diseases such aspsoriasis, psoriatic arthritis, rheumatoid arthritis, endotoxemia,inflammatory bowel disease (IBD), IBS, colitis, asthma, allograftrejection, immune mediated renal diseases, hepatobiliary diseases,multiple sclerosis; atherosclerosis, promotion of tumor growth, ordegenerative joint disease and other inflammatory conditions disclosedherein.

Within preferred embodiments, the antibodies of the invention bind to,blocks, inhibits, reduces, antagonizes or neutralizes IL-17F and IL-17A(individually or together) in vivo.

Thus, particular embodiments of the present invention are directedtoward use of the antibodies of the invention as antagonists ininflammatory and immune diseases or conditions such as psoriasis,psoriatic arthritis, atopic dermatitis, inflammatory skin conditions,rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's Disease,diverticulosis, asthma, pancreatitis, type I diabetes (IDDM), pancreaticcancer, pancreatitis, Graves Disease, colon and intestinal cancer,autoimmune disease, sepsis, organ or bone marrow transplant;inflammation due to endotoxemia, trauma, surgery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells), suppression ofimmune response to a pathogen or antigen, or other instances whereinhibition of IL-17F and IL-17A cytokines is desired.

Moreover, the antibodies of the invention are useful to:

(1) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A and IL-17F in the treatment of acute inflammation, inflammationas a result of trauma, tissue injury, surgery, sepsis or infection, andchronic inflammatory diseases such as asthma, inflammatory bowel disease(IBD), IBS, chronic colitis, splenomegaly, rheumatoid arthritis,recurrent acute inflammatory episodes (e.g., tuberculosis), andtreatment of amyloidosis, and atherosclerosis, Castleman's Disease,asthma, and other diseases associated with the induction of acute-phaseresponse.

(2) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A or IL-17F in the treatment of autoimmune diseases such as IDDM,multiple sclerosis (MS), systemic Lupus erythematosus (SLE), myastheniagravis, rheumatoid arthritis, IBS and IBD to prevent or inhibitsignaling in immune cells (e.g. lymphocytes, monocytes, leukocytes) viatheir receptors (e.g. IL-17RA and IL-17RC). Blocking, inhibiting,reducing, or antagonizing signaling via IL-17RA and IL-17RC, using theantibodies of the present invention, may also benefit diseases of thepancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,pancreatitis, and pancreatic carcinoma may benefit. Mabs to IL-17A andIL-17F may also be useful to treat nephropathies such asglomerulosclerosis, membranous neuropathy, amyloidosis (which alsoaffects the kidney among other tissues), renal arteriosclerosis,glomerulonephritis of various origins, fibroproliferative diseases ofthe kidney, as well as kidney dysfunction associated with SLE, IDDM,type II diabetes (NIDDM), renal tumors and other diseases.

(3) Agonize, enhance, increase or initiate signaling via IL-17A orIL-17F receptors in the treatment of autoimmune diseases such as IDDM,MS, SLE, myasthenia gravis, rheumatoid arthritis, IBS, and IBD.Anti-IL-17A and IL-17F neutralizing and monoclonal antibodies may signallymphocytes or other immune cells to differentiate, alter proliferation,or change production of cytokines or cell surface proteins thatameliorate autoimmunity. Specifically, modulation of a T-helper cellresponse to an alternate pattern of cytokine secretion may deviate anautoimmune response to ameliorate disease (Smith J A et al., J. Immunol.160:4841-4849, 1998). Similarly, agonistic antibodies may be used tosignal, deplete and deviate immune cells involved in asthma, allergy andatopic disease. Signaling via IL-17RA and IL-17RC may also benefitdiseases of the pancreas, kidney, pituitary and neuronal cells. IDDM,NIDDM, pancreatitis, and pancreatic carcinoma may benefit.

The antibodies described herein can be used to bind, block, inhibit,reduce, antagonize or neutralize IL-17F and IL-17A activity, eithersingly or together, in the treatment of autoimmune disease, atopicdisease, NIDDM, pancreatitis and kidney dysfunction as described above.The antibodies of the present invention are useful as antagonists ofIL-17A or IL-17F cytokine. Such antagonistic effects can be achieved bydirect neutralization or binding of IL-17A and IL-17F.

Inflammation is a protective response by an organism to fend off aninvading agent. Inflammation is a cascading event that involves manycellular and humoral mediators. On one hand, suppression of inflammatoryresponses can leave a host immunocompromised; however, if leftunchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., psoriasis, arthritis, rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease and the like),septic shock and multiple organ failure. Importantly, these diversedisease states share common inflammatory mediators. The collectivediseases that are characterized by inflammation have a large impact onhuman morbidity and mortality. Therefore it is clear that the antibodiesof the present invention could have crucial therapeutic potential for avast number of human and animal diseases, from asthma and allergy toautoimmunity and septic shock.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use of antibodiesthat antagonize, neutralize or block both IL-17A and IL-17F. Forexample, rheumatoid arthritis (RA) is a systemic disease that affectsthe entire body and is one of the most common forms of arthritis. It ischaracterized by the inflammation of the membrane lining the joint,which causes pain, stiffness, warmth, redness and swelling. Inflammatorycells release enzymes that may digest bone and cartilage. As a result ofrheumatoid arthritis, the inflamed joint lining, the synovium, caninvade and damage bone and cartilage leading to joint deterioration andsevere pain amongst other physiologic effects. The involved joint canlose its shape and alignment, resulting in pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002). Oneof those mediators could be IL-17A or IL-17F, and as such a moleculethat binds or inhibits IL-17F or IL-17A activity, such as solubleIL-17RA, IL-17RA polypeptides, or anti IL-17RA antibodies or bindingpartners, could serve as a valuable therapeutic to reduce inflammationin rheumatoid arthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995).

One group has shown that an anti-mouse IL-17 antibody reduces symptomsin a mouse CIA-model relative to control mice, thus showing conceptuallythat an anti-IL-17A antibody may be beneficial in treating humandisease, as the administration of a single mouse-IL-17-specific ratantisera reduced the symptoms of arthritis in the animals whenintroduced prophylactically or after symptoms of arthritis were alreadypresent in the model (Lubberts et al, Arthritis Rheum. 50:650-9, 2004).Therefore, an antibody that antagonizes, neutralizes or blocks IL-17Aand IL-17F binding to their respective receptors can be used toneutralize IL-17A and/or IL-17F in the treatment of specific humandiseases such as arthritis, psoriasis, psoriatic arthritis, endotoxemia,inflammatory bowel disease (IBD), IBS, colitis, and other inflammatoryconditions disclosed herein.

The administration of antibodies of the invention to these CIA modelmice is used to evaluate the use of such an antibody as an antagonist toIL-17A and IL-17F, which could be used to ameliorate symptoms and alterthe course of disease. By way of example and without limitation, theinjection of 10-200 μg of an antibody of the present invention per mouse(one to seven times a week for up to but not limited to 4 weeks vias.c., i.p., or i.m route of administration) can significantly reduce thedisease score (paw score, incident of inflammation, or disease).Depending on the initiation of antibody administration (e.g. prior to orat the time of collagen immunization, or at any time point following thesecond collagen immunization, including those time points at which thedisease has already progressed), such anti-IL-17A and IL-17F antibodiescan be efficacious in preventing rheumatoid arthritis, as well aspreventing its progression.

2. Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory proteins, such as antibodies of the invention, couldaid in preventing and treating endotoxemia in humans and animals. Suchantibodies could serve as a valuable therapeutic to reduce inflammationand pathological effects in endotoxemia.

Lipopolysaccharide (LPS) induced endotoxemia engages many of theproinflammatory mediators that produce pathological effects in theinfectious diseases and LPS induced endotoxemia in rodents is a widelyused and acceptable model for studying the pharmacological effects ofpotential pro-inflammatory or immunomodulating agents. LPS, produced ingram-negative bacteria, is a major causative agent in the pathogenesisof septic shock (Glausner et al., Lancet 338:732, 1991). A shock-likestate can indeed be induced experimentally by a single injection of LPSinto animals. Molecules produced by cells responding to LPS can targetpathogens directly or indirectly. Although these biological responsesprotect the host against invading pathogens, they may also cause harm.Thus, massive stimulation of innate immunity, occurring as a result ofsevere Gram-negative bacterial infection, leads to excess production ofcytokines and other molecules, and the development of a fatal syndrome,septic shock syndrome, which is characterized by fever, hypotension,disseminated intravascular coagulation, and multiple organ failure(Dumitru et al. Cell 103:1071-1083, 2000).

These toxic effects of LPS are mostly related to macrophage activationleading to the release of multiple inflammatory mediators. Among thesemediators, TNF appears to play a crucial role, as indicated by theprevention of LPS toxicity by the administration of neutralizinganti-TNF antibodies (Beutler et al., Science 229:869, 1985). It is wellestablished that 1 ug injection of E. coli LPS into a C57Bl/6 mouse willresult in significant increases in circulating IL-6, TNF-alpha, IL-1,and acute phase proteins (for example, SAA) approximately 2 hours postinjection. The toxicity of LPS appears to be mediated by these cytokinesas passive immunization against these mediators can result in decreasedmortality (Beutler et al., Science 229:869, 1985). The potentialimmunointervention strategies for the prevention and/or treatment ofseptic shock include anti-TNF mAb, IL-1 receptor antagonist, LIF, IL-10,and G-CSF.

The administration of antibodies of the invention to these LPS-inducedmodel may be used to evaluate the use of such antibodies to amelioratesymptoms and alter the course of LPS-induced disease. Moreover, resultsshowing inhibition of IL-17A and IL-17F by antibodies of the inventionprovide proof of concept that such antibodies can also be used toameliorate symptoms in the LPS-induced model and alter the course ofdisease. The model will show induction of IL-17A and IL-17F by LPSinjection and the potential treatment of disease by such antibodies.Since LPS induces the production of pro-inflammatory factors possiblycontributing to the pathology of endotoxemia, the neutralization of bothIL-17A and IL-17F activity or other pro-inflammatory factors byantibodies of the invention can be used to reduce the symptoms ofendotoxemia, such as seen in endotoxic shock.

3. Inflammatory Bowel Disease IBD

In the United States approximately 500,000 people suffer fromInflammatory Bowel Disease (IBD) which can affect either colon andrectum (Ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. Antibodies thatbind both IL-17A and IL-17F could serve as a valuable therapeutic toreduce inflammation and pathological effects in IBD and relateddiseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers formreleasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (e.g.,azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of antibodies of the invention to these TNBS or DSSmodels can be used to evaluate the use such antibodies to amelioratesymptoms and alter the course of gastrointestinal disease. Moreover, theresults showing inhibition of IL-17A and IL-17F by such antibodiesprovide proof of concept that antibodies of the invention can also beused to ameliorate symptoms in the colitis/IBD models and alter thecourse of disease.

4. Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. Antibodies thatbind both IL-17A and IL-17F could serve as a valuable therapeutic toreduce inflammation and pathological effects in psoriasis, otherinflammatory skin diseases, skin and mucosal allergies, and relateddiseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

Antibodies that bind IL-17A and IL-17F may also be used withindiagnostic systems for the detection of circulating levels of IL-17F orIL-17A, and in the detection of IL-17A and/or IL-17F associated withacute phase inflammatory response. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including inflammation or cancer. IL-17A and IL-17F areknown to induce associated acute phase inflammatory response. Moreover,detection of acute phase proteins or molecules such as IL-17A or IL-17Fcan be indicative of a chronic inflammatory condition in certain diseasestates (e.g., asthma, psoriasis, rheumatoid arthritis, colitis, IBD).Detection of such conditions serves to aid in disease diagnosis as wellas help a physician in choosing proper therapy.

In addition to other disease models described herein, the activity ofantibodies of the invention on inflammatory tissue derived from humanpsoriatic lesions can be measured in vivo using a severe combined immunedeficient (SCID) mouse model. Several mouse models have been developedin which human cells are implanted into immunodeficient mice(collectively referred to as xenograft models); see, for example, CattanA R, Douglas E, Leuk. Res. 18:513-22, 1994 and Flavell, D J,Hematolonical Oncology 14:67-82, 1996. As an in vivo xenograft model forpsoriasis, human psoriatic skin tissue is implanted into the SCID mousemodel, and challenged with an appropriate antagonist. Moreover, otherpsoriasis animal models in the art may be used to evaluate theantibodies of the invention, such as human psoriatic skin graftsimplanted into AGR129 mouse model, and challenged with an appropriateantagonist (e.g., see, Boyman, O. et al., J. Exp. Med. Onlinepublication #20031482, 2004, incorporated herein by reference).Anti-IL-17A and IL-17F antibodies that bind, block, inhibit, reduce,antagonize or neutralize the activity of IL-17A and IL-17F are preferredantagonists. Similarly, tissues or cells derived from human colitis,IBD, arthritis, or other inflammatory lesions can be used in the SCIDmodel to assess the anti-inflammatory properties of the antibodies ofthe invention described herein.

Therapies designed to abolish, retard, or reduce inflammation usingantibodies of the invention can be tested by administration of suchantibodies to SCID mice bearing human inflammatory tissue (e.g.,psoriatic lesions and the like), or other models described herein.Efficacy of treatment is measured and statistically evaluated asincreased anti-inflammatory effect within the treated population overtime using methods well known in the art. Some exemplary methodsinclude, but are not limited to measuring for example, in a psoriasismodel, epidermal thickness, the number of inflammatory cells in theupper dermis, and the grades of parakeratosis. Such methods are known inthe art and described herein. For example, see Zeigler, M. et al. LabInvest 81:1253, 2001; Zollner, T. M. et al. J. Clin. Invest. 109:671,2002; Yamanaka, N. et al. Microbio.l Immunol. 45:507, 2001;Raychaudhuri, S. P. et al. Br. J. Dermatol. 144:931, 2001; Boehncke, W.H et al. Arch. Dermatol. Res. 291:104, 1999; Boehucke, W. H et al. J.Invest. Dennatol. 116:596, 2001; Nickoloff, B. J. et al. Am. J. Pathol.146:580, 1995; Boehncke, W. H et al. J. Cutan. Pathol. 24:1, 1997;Sugai, J., M. et al. J. Dermatol. Sci. 17:85, 1998; and Villadsen L. S.et al. J. Clin. Invest. 112:1571, 2003. Inflammation may also bemonitored over time using well-known methods such as flow cytometry (orPCR) to quantitate the number of inflammatory or lesional cells presentin a sample, score (weight loss, diarrhea, rectal bleeding, colonlength) for IBD, paw disease score and inflammation score for CIA RAmodel.

Moreover, Psoriasis is a chronic inflammatory skin disease that isassociated with hyperplastic epidermal keratinocytes and infiltratingmononuclear cells, including CD4+ memory T cells, neutrophils andmacrophages (Christophers, Int. Arch. Allergy Immunol., 110.199, 1996).It is currently believed that environmental antigens play a significantrole in initiating and contributing to the pathology of the disease.However, it is the loss of tolerance to self-antigens that is thought tomediate the pathology of psoriasis. Dendritic cells and CD4⁺ T cells arethought to play an important role in antigen presentation andrecognition that mediate the immune response leading to the pathology.We have recently developed a model of psoriasis based on the CD4+CD45RBtransfer model (Davenport et al., Internat. Immunopharmacol.,2:653-672). Antibodies of the present invention are administered to themice. Inhibition of disease scores (skin lesions, inflammatorycytokines) indicates the effectiveness of such antibodies in psoriasis.

5. Atopic Dermatitis.

AD is a common chronic inflammatory disease that is characterized byhyperactivated cytokines of the helper T cell subset 2 (Th2). Althoughthe exact etiology of AD is unknown, multiple factors have beenimplicated, including hyperactive Th2 immune responses, autoimmunity,infection, allergens, and genetic predisposition. Key features of thedisease include xerosis (dryness of the skin), pruritus (itchiness ofthe skin), conjunctivitis, inflammatory skin lesions, Staphylococcusaureus infection, elevated blood eosinophilia, elevation of serum IgEand IgG1, and chronic dermatitis with T cell, mast cell, macrophage andeosinophil infiltration. Colonization or infection with S. aureus hasbeen recognized to exacerbate AD and perpetuate chronicity of this skindisease.

AD is often found in patients with asthma and allergic rhinitis, and isfrequently the initial manifestation of allergic disease. About 20% ofthe population in Western countries suffer from these allergic diseases,and the incidence of AD in developed countries is rising for unknownreasons. AD typically begins in childhood and can often persist throughadolescence into adulthood. Current treatments for AD include topicalcorticosteroids, oral cyclosporin A, non-corticosteroidimmunosuppressants such as tacrolimus (FK506 in ointment form), andinterferon-gamma. Despite the variety of treatments for AD, manypatients' symptoms do not improve, or they have adverse reactions tomedications, requiring the search for other, more effective therapeuticagents. The antibodies of the present invention, including theneutralizing anti-human IL-17A and IL-17F antibodies of the presentinvention, can be used to neutralize IL-17F and IL-17A in the treatmentof specific human diseases such as atoptic dermatitis, inflammatory skinconditions, and other inflammatory conditions disclosed herein.

6. Asthma

IL-17 plays an important role in allergen-induced T cell activation andneutrophilic influx in the airways. The receptor for IL-17 is expressedin the airways (Yao, et al. Immunity 3:811 (1995)) and IL-17 mediatedneutrophil recruitment in allergic asthma is largely induced by thechemoattractant IL-8, GRO-α and macrophage inflammatory protein-2(MIP-2) produced by IL-17 stimulated human bronchial epithelial cells(HBECs) and human bronichial fibroblasts (Yao, et al. J Immunol 155:5483(1995)); Molet, et al. J Allergy Clin Immunol 108:430 (2001)). IL-17also stimulates HBECs to release IL-6, a neutrophil-activating factor(Fossiez, et al, J Exp Med 183:2593 (1996), and Linden, et al. Int ArchAllergy Immunol 126:179 (2001)) and has been shown to synergize withTNF-α to prolong the survival of human neutrophils in vitro (Laan, etal. Eur Respir J 21:387 (2003)). Moreover, IL-17 is capable ofamplifying the inflammatory responses in asthma by its ability toenhance the secretion of cytokines implicated in airway remodeling suchas the profibrotic cytokines, IL-6 and IL-1 and inflammatory mediatorsgranulocyte colony-stimulating factor (G-CSF) and granulocyte macrophagecolony-stimulating factor (GM-CSF) (Molet, et al. J Allergy Clin Immunol108:430 (2001)).

Clinical evidence shows that acute, severe exacerbations of asthma areassociated with recruitment and activation of neutrophils in theairways, thus IL-17 is likely to play a significant role in asthma.Patients with mild asthma display a detectable increase in the localconcentration of free, soluble IL-17A protein (Molet, et al. J AllergyClin Immunol 108:430 (2001)) while healthy human volunteers withinduced, severe airway inflammation due to the exposure to a swineconfinement, display a pronounced increase in the concentration of free,soluble IL-17A protein in the bronchioalveolar space (Fossiez, et al, JExp Med 183:2593 (1996), and Linden, et al. Int Arch Allergy Immunol126:179 (2001)). Furthermore, IL-17 levels in sputum have correlatedwith individuals who have increased airway hyper-reactivity Barczyk, etal. Respir Med 97:726 (2003).

In animal models of airway hyper-responsiveness, chronic inhalation ofovalbumin by sensitized mice resulted in bronchial eosinophilicinflammation and early induction of IL-17 mRNA expression in inflamedlung tissue, together with a bronchial neutrophilia Hellings, et al. AmJ Respir Cell Mol Biol 28:42 (2003). Anti-IL-17 monoclonal antibodiesstrongly reduced bronchial neutrophilic influx but significantlyenhanced IL-5 levels in both bronchioalveolar lavage fluid and serum,and aggravated allergen-induced bronchial eosinophilic influx,suggesting that IL-17A may be involved in determining the balancebetween neutrophil and eosinophil accumulation following antigen insultId.

Among the IL-17 family members, IL-17F is most closely related toIL-17A. The biological activities mediated by IL-17F are similar tothose of IL-17A, where IL-17F stimulates production of IL-6, IL-8 andG-CSF Hurst, et al. J Immunol 169:443 (2002). IL-17F also inducesproduction of IL-2, transforming growth factor (TGF)-α, and monocytechemoattractant protein (MCP) in endothelial cells Starnes, et al. JImmunol 167:4137 (2001). Similarly, allergen challenge can increaselocal IL-17F in patients with allergic asthma Kawaguchi, et al. JImmunol 167:4430 (2001). Gene delivery of IL-17F in murine lungincreases neutrophils in the bronchioalveolar space, while mucosaltransfer of the IL-17F gene enhances the levels of Ag-induced pulmonaryneutrophilia and airway responsiveness to methacholine Oda, et al. Am JRespir Crit. Care Med 171:12 (2005).

Apart from asthma, several chronic inflammatory airway diseases arecharacterized by neutrophil recruitment in the airways and IL-17 hasbeen reported to play an important role in the pathogenesis ofrespiratory conditions such as chronic obstructive pulmonary disease(COPD), bacterial pneumonia and cystic fibrosis (Linden, et al. EurRespir J 15:973 (2000), Ye, et al. Am J Respir Cell Mol Biol 25:335(2001), Rahman, et al. Clin Immunol 115:268 (2005)). An anti-IL-17A andanti-IL-17F antibody could be demonstrated to be efficacious for chronicinflammatory airway disease in an in vitro model of inflammation. Theability of antagonists to IL-17F and IL-17A activity, such as theantibodies of the present invention, to inhibit IL-17A or andIL-17F-induced cytokine and chemokine production from cultured HBECs orbronchial fibroblasts could be used as a measure of efficacy for suchantagonists in the prevention of the production of inflammatorymediators directly resulting from IL-17A and/or F stimulation. If theaddition of antagonists to IL-17F and IL-17A activity, such as theantibodies of the invention markedly reduces the production andexpression of inflammatory mediators, it would be expected to beefficacious in inflammatory aspects associated with chronic airwayinflammation.

7. Irritable Bowel Syndrome (“IBS”)

Irritable bowel syndrome represents a disease characterized by abdominalpain or discomfort and an erratic bowel habit. IBS patients can becharacterized into three main groups based on bowel habits: those withpredominantly loose or frequent stools, those with predominantly hard orinfrequent stools, and those with variable or normal stools (Talley etal., 2002). Altered intestinal motility, abnormalities in epithelialfunction, abnormal transit of stool and gas, and stress, may contributeto symptoms, while visceral hypersensitivity is a key feature in mostpatients. Genetic factors affecting pain-signaling and disturbances incentral processing of afferent signals are postulated to predisposeindividuals to IBS following specific environmental exposures. Studieshave also demonstrated that inflammatory responses in the colon maycontribute to increased sensitivity of smooth muscle and enteric nervesand therefore perturb sensory-motor functions in the intestine (Collinset al., 2001). There is clinical overlap between IBS and IBD, withIBS-like symptoms frequently reported in patients before the diagnosisof IBD, and a higher than expected IBS symptoms in patients in remissionfrom established IBD. Thus, these conditions may coexist with a higherthan expected frequency, or may exist on a continuum, with IBS and IBDat different ends of the same spectrum. However, it should be noted thatin most IBS patients, colonic biopsy specimens appear normal.Nevertheless, IBS significantly affects a very large number ofindividuals (U.S. prevalence in 2000, approximately 16 millionindividuals), resulting in a total cost burden of 1.7 billion dollars(year 2000). Thus, among the most prevalent and costly gastrointestinaldiseases and disorders, IBS is second only to gastroesophageal refluxdisease (GERD). Yet unlike GERD, treatment for IBS remainsunsatisfactory (Talley et al., 2002; Farhadi et al., 21001; Collins etal., 2001), demonstrating that IBS clearly represents an unmet medicalneed.

Converging disease models have been proposed that postulate an enhancedresponsiveness of neural, immune or neuroimmune circuits in the centralnervous system (CNS) or in the gut to central (psychosocial) orperipheral (tissue irritation, inflammation, infection) perturbations ofnormal homeostasis (Talley et al., 2002). This enhanced responsivenessresults in dysregulation of gut motility, epithelial function (immune,permeability), and visceral hypersensitivity, which in turn results inIBS symptoms.

There may be a role for a number of different molecules in thepathogenesis of IBS including a role for molecules that stimulateneurons and those that are involved in initiation of inflammatoryprocess. A number of molecules are known to be linked to possibleactivity on neurons due to their direct expression by neurons orexpression of their receptors on neurons, including IL-17D, IL-17B andIL-31. Moreover, a number of IL-17 family members and related moleculeshave been associated with inflammation in the gut, including IL-17A,IL-17F, IL-23 and IL-31.

Efficacy of inhibitors/antagonists of these molecules could be tested invivo in animal models of disease. Several animal models have beenproposed that mimic key features of IBS and involve centrally targetedstimuli (stress) or peripherally targeted stimuli (infection,inflammation). Two examples of in vivo animal models that can be used todetermine the effectiveness of inhibitors in the treatment of IBS are(i) models focusing on primary CNS-directed pathogenesis of IBS (stressmodels), and (ii) models focusing on gut-directed inducers of stress(i.e. gut inflammation, infection or physical stress). It should benoted however, that events within the CNS or in the gastrointestinal(GI) tract do not occur in isolation and that symptoms of IBS mostlikely result from a complex interaction between signals from the CNS onthe GI and vice versa.

For pharmaceutical use, the antibodies of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection, controlled release, e.g, using mini-pumps orother appropriate technology, or by infusion over a typical period ofone to several hours. In general, pharmaceutical formulations willinclude a hematopoietic protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. When utilizing such acombination therapy, the cytokines may be combined in a singleformulation or may be administered in separate formulations. Methods offormulation are well known in the art and are disclosed, for example, inRemington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton Pa., 1990, which is incorporated herein by reference. Therapeuticdoses will generally be in the range of 0.1 to 100 mg/kg of patientweight per day, preferably 0.5-20 mg/kg per day, with the exact dosedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. The proteins will commonly be administered over a period ofup to 28 days following chemotherapy or bone-marrow transplant or untila platelet count of >20,000/mm³, preferably >50,000/mm³, is achieved.More commonly, the proteins will be administered over one week or less,often over a period of one to three days. In general, a therapeuticallyeffective amount of antibodies of the present invention is an amountsufficient to produce a clinically significant increase in theproliferation and/or differentiation of lymphoid or myeloid progenitorcells, which will be manifested as an increase in circulating levels ofmature cells (e.g. platelets or neutrophils). Treatment of plateletdisorders will thus be continued until a platelet count of at least20,000/mm³, preferably 50,000/mm³, is reached. The antibodies of thepresent invention can also be administered in combination with othercytokines such as IL-3, -6 and -11; stem cell factor; erythropoietin;G-CSF and GM-CSF. Within regimens of combination therapy, daily doses ofother cytokines will in general be: EPO, 150 U/kg; GM-CSF, 5-Ig/kg;IL-3, 1-5 lg/kg; and G-CSF, 1-25 lg/kg. Combination therapy with EPO,for example, is indicated in anemic patients with low EPO levels.

Generally, the dosage of administered antibodies will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibodies which isin the range of from about 1 pg/kg to 10 mg/kg (amount of agent/bodyweight of patient), although a lower or higher dosage also may beadministered as circumstances dictate.

Administration of antibodies of the invention to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingantibodies of the invention can be prepared and inhaled with the aid ofdry-powder dispersers, liquid aerosol generators, or nebulizers (e.g.,Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. DrugDeliv. Rev, 35:235 (1999)). This approach is illustrated by the AERXdiabetes management system, which is a hand-held electronic inhaler thatdelivers aerosolized insulin into the lungs. Studies have shown thatproteins as large as 48,000 kDa have been delivered across skin attherapeutic concentrations with the aid of low-frequency ultrasound,which illustrates the feasibility of transcutaneous administration(Mitragotri et al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingIL-17A and IL-17F binding activity (Potts et al., Pharm. Biotechnol.10:213 (1997)).

A pharmaceutical composition comprising an antibodies of the inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the therapeutic proteins are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient patient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, antibodies of the invention and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a therapeuticmolecule of the present invention and a pharmaceutically acceptablecarrier is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. For example,an agent used to treat inflammation is physiologically significant ifits presence alleviates the inflammatory response. Effective treatmentmay be assessed in a variety of ways. In one embodiment, effectivetreatment is determined by reduced inflammation. In other embodiments,effective treatment is marked by inhibition of inflammation. In stillother embodiments, effective therapy is measured by increased well-beingof the patient including such signs as weight gain, regained strength,decreased pain, thriving, and subjective indications from the patient ofbetter health.

A pharmaceutical composition comprising antibodies of the invention canbe furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions and oral suspensions.Exemplary solid forms include capsules, tablets, and controlled-releaseforms. The latter form is illustrated by miniosmotic pumps and implants(Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 95-123 (CRC Press 1995); Bremer et al., “Protein Delivery withInfusion Pumps,” in Protein Delivery: Physical Systems, Sanders andHendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al.,“Delivery of Proteins from a Controlled Release Injectable Implant,” inProtein Delivery: Physical Systems, Sanders and Hendren (eds.), pages93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. Clin. Microbiol. Infect.Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), and Ranade,“Site-Specific Drug Delivery Using Liposomes as Carriers,” in DrugDelivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRC Press1995)). Liposomes are similar in composition to cellular membranes andas a result, liposomes can be administered safely and are biodegradable,Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.02 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s) (see,for example, Machy et al., Liposomes In Cell Biology And Pharmacology(John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576(1989)). Moreover, it is possible to control the therapeuticavailability of the encapsulated agent by varying liposome size, thenumber of bilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides and antibodies can be encapsulated within liposomes usingstandard techniques of protein microencapsulation (see, for example,Anderson et al., Infect. Immun. 31:1099 (1981), Anderson et al., CancerRes. 50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology, 2nd Edition, Vol. III, Gregoriadis(ed.), page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnot 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding IL-17A and IL-17F activity such as anti-IL-17A and IL-17Fantibodies, which a polypeptide is linked with a polymer, as discussedabove.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises an antibody of the invention.Antibodies of the invention can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the antibody composition is contraindicated in patientswith known hypersensitivity to IL-17A and IL-17F.

A pharmaceutical composition comprising antibodies of the invention canbe furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions, aerosols, droplets,topological solutions and oral suspensions. Exemplary solid formsinclude capsules, tablets, and controlled-release forms. The latter formis illustrated by miniosmotic pumps and implants (Bremer et al., Pharm.Biotechnol. 10:239 (1997); Ranade, “Implants in Drug Delivery,” in DrugDelivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press1995); Bremer et al., “Protein Delivery with Infusion Pumps,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254(Plenum Press 1997); Yewey et al., “Delivery of Proteins from aControlled Release Injectable Implant,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).Other solid forms include creams, pastes, other topologicalapplications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur J. Clin. Microbio.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies of the present invention, antibodyfragments, carbohydrates, vitamins, and transport proteins. For example,liposomes can be modified with branched type galactosyllipid derivativesto target asialoglycoprotein (galactose) receptors, which areexclusively expressed on the surface of liver cells (Kato and Sugiyama,Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al.,Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology27:772 (1998), have shown that labeling liposomes with asialofetuin ledto a shortened liposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

The antibodies of the invention can be encapsulated within liposomesusing standard techniques of protein microencapsulation (see, forexample, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

The present invention contemplates compositions of antibodies, whereinsaid antibodies bind to both IL-17A and IL-17F, and methods andtherapeutic uses comprising an such an antibody as described herein.Such compositions can further comprise a carrier. The carrier can be aconventional organic or inorganic carrier. Examples of carriers includewater, buffer solution, alcohol, propylene glycol, macrogol, sesame oil,corn oil, and the like.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 IL-17F mRNA is Upregulated in a Murine Model ofAsthma

IL-17F mRNA levels were measured in a sensitization and airway challengemodel in mice. Groups of mice, 8 to 10 wks of age, were sensitized byintraperitoneal injection of 10 ug of recombinant Dermatophagoidespteronyssinus allergen 1 (DerP1) (Indoor biotechnologies, Cardiff, UK)in 50% Imject Alum (Pierce) on days 0 and 7. Seven days later, mice werechallenged on 3 consecutive days (days 14, 15 and 16) with 20 ug ofDerP1 in 50 ul PBS. There were 4 mice representing this group. Negativecontrols included 5 mice given phosphate buffered saline (PBS)sensitization, followed by PBS challenge. In addition to 3 mice givenDerP1 sensitization, followed by PBS challenge. Forty-eight hoursfollowing allergen, or control challenge whole lung tissue was harvestedand total RNA was isolated.

First strand cDNA was prepared using identical amounts of total RNA fromeach subject. IL-17F PCR was applied using Qiagen hotstar polymerase(Qiagen, Valencia, Calif.) and the manufacturer's recommendations. TheIL-17F PCR utilized 35 cycles of amplification with sense primer,zc46098 (SEQ ID NO:9) and antisense primer, zc46099 (SEQ ID NO:10). Inorder to establish that the template quality was uniform amongst allsubjects, Beta Actin PCR was applied to the same amount of each templateused in the IL-17F amplification. B actin PCR included 25 cycles of PCRwith sense primer, zc44779 (SEQ ID NO:11) and antisense primer, zc44776(SEQ ID N:12).

All 4 mice from the DerP1 sensitized, DerP1 challenged treatment group(the asthma simulation) showed robust IL-17F amplification. In contrast,weak IL-17F amplification was seen from the negative controls, including3 of 3 subjects representing the DerP1 sensitized/PBS challengedtreatment group and 5 of 5 subjects from the PBS sensitized/PBSchallenged treatment group. B actin amplification was at least as robustfor the negative controls as for the asthma-simulated subjects,demonstrating that the weak negative control IL-17F amplification wasnot due to template problems.

Example 2 IL-17A Induces Elevated Levels of IFN-Gamma and TNF-Alpha inHuman Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMC) are purified by ficolldensity gradient centrifugation and then incubated overnight at 37° C.in media alone, 50 ng/ml anti-human CD3 antibody, or the combination of50 ng/ml anti-human CD3 antibody plus 1 μg/ml anti-human CD28 antibody.Replicate cultures for each of these conditions are set up and are givenno cytokine, ng/ml human IL-17A, or 25 ng/ml human IL-17F. After 24-hourincubations, supernatants from each culture are harvested and assayedfor cytokine content using B-D Bioscience's human Th1/Th2 CytometricBead Array (CBA). We found that cultures that had been stimulated witheither anti-CD3 or anti-CD3 plus anti-CD28 and had been supplementedwith IL-17A contained significantly elevated levels of IFN-gamma andTNF-alpha (3-5-fold elevation of each) over cultures with no cytokineadded or those that received IL-17F. Cultures in which no anti-CD3stimulation was added did not show significant changes in cytokinelevels. In addition, IL-17A addition induced no significant changes inother cytokines assayed for with the CBA including IL-2, IL-4, IL-5, andIL-10. This data indicates that IL-17A, but not IL-17F, can augment theproduction of IFN-gamma and TNF-alpha in PBMC cultures stimulated withanti-CD3 or anti-CD3 plus anti-CD28.

Example 3 An Anti-IL-17A and IL-17F Antibody Decreases Disease Incidenceand Progression in Mouse Collagen Induced Arthritis (CIA) Model A) MouseCollagen Induced Arthritis (CIA) Model

Ten week old male DBA/1J mice (Jackson Labs) are divided into 3 groupsof 13 mice/group. On day-21, animals are given an intradermal tailinjection of 50-100 μl of 1 mg/ml chick Type II collagen formulated inComplete Freund's Adjuvant (prepared by Chondrex, Redmond, Wash.), andthree weeks later on Day 0 they are given the same injection exceptprepared in Incomplete Freund's Adjuvant. An antibody of the invention(e.g. a cross-reactive antibody or a bispecific antibody) isadministered as an intraperitoneal injection 3 times a week for 4 weeks,at different time points ranging from Day 0, to a day in which themajority of mice exhibit moderate symptoms of disease. Groups receiveeither 10 or 100 j±g of the antibody per animal per dose, and controlgroups receive the vehicle control, PBS (Life Technologies, Rockville,Md.). Animals begin to show symptoms of arthritis following the secondcollagen injection, with most animals developing inflammation within1.5-3 weeks. The extent of disease is evaluated in each paw by using acaliper to measure paw thickness, and by assigning a clinical score(0-3) to each paw: 0=Normal, 0.5=Toe(s) inflamed, 1=Mild pawinflammation, 2=Moderate paw inflammation, and 3=Severe paw inflammationas detailed below.

B) Monitoring Disease

Animals can begin to show signs of paw inflammation soon after thesecond collagen injection, and some animals may even begin to have signsof toe inflammation prior to the second collagen injection. Most animalsdevelop arthritis within 1.5-3 weeks of the boost injection, but somemay require a longer period of time. Incidence of disease in this modelis typically 95-100%, and 0-2 non-responders (determined after 6 weeksof observation) are typically seen in a study using 40 animals. Notethat as inflammation begins, a common transient occurrence of variablelow-grade paw or toe inflammation can occur. For this reason, an animalis not considered to have established disease until marked, persistentpaw swelling has developed.

All animals are observed daily to assess the status of the disease intheir paws, which is done by assigning a qualitative clinical score toeach of the paws. Every day, each animal has its 4 paws scored accordingto its state of clinical disease. To determine the clinical score, thepaw can be thought of as having 3 zones, the toes, the paw itself (manusor pes), and the wrist or ankle joint. The extent and severity of theinflammation relative to these zones is noted including: observation ofeach toe for swelling; torn nails or redness of toes; notation of anyevidence of edema or redness in any of the paws; notation of any loss offine anatomic demarcation of tendons or bones; evaluation of the wristor ankle for any edema or redness; and notation if the inflammationextends proximally up the leg. A paw score of 1, 2, or 3 is based firston the overall impression of severity, and second on how many zones areinvolved. The scale used for clinical scoring is shown below.

C) Clinical Score

-   -   0=Normal    -   0.5=One or more toes involved, but only the toes are inflamed    -   1=mild inflammation involving the paw (1 zone), and may include        a toe or toes    -   2=moderate inflammation in the paw and may include some of the        toes and/or the wrist/ankle (2 zones)    -   3=severe inflammation in the paw, wrist/ankle, and some or all        of the toes (3 zones)

Established disease is defined as a qualitative score of pawinflammation ranking 2 or more, that persists for two days in a row.Once established disease is present, the date is recorded and designatedas that animal's first day with “established disease”.

Blood is collected throughout the experiment to monitor serum levels ofanti-collagen antibodies, as well as serum immunoglobulin and cytokinelevels. Serum anti-collagen antibodies correlate well with severity ofdisease. Animals are euthanized on Day 21, and blood collected for serumand CBC's. From each animal, one affected paw is collected in 10% NBFfor histology and one is frozen in liquid nitrogen and stored at −80° C.for mRNA analysis. Also, ½ spleen, ½ thymus, ½ mesenteric lymph node,one liver lobe and the left kidney are collected in RNAlater for RNAanalysis, and ½ spleen, ½ thymus, ½ mesenteric lymph node, the remainingliver, and the right kidney are collected in 10% NBF for histology.Serum is collected and frozen at −80° C. for immunoglobulin and cytokineassays.

Groups of mice receiving an antibody of the invention at all time pointsare characterized by a delay in the onset and/or progression of pawinflammation. These results indicate that an IL-17A and IL-17Fcross-reactive and/or bispecific antibody can reduce inflammation, aswell as disease incidence and progression associated with this model.These results are further supported by the observation thatadministration of such antibody resulted in decreased levels of serumTNFa, IL-1b, and anti-collagen antibodies.

Example 4 An Anti-IL-17A and IL-17F Antibody Decreases Disease Incidenceand Progression in an Inflammatory Bowel Disease (IBD) Model

This model is designed to show that cultured intestinal tissue frompatients with IBD produce higher levels of inflammatory mediatorscompared to tissue from healthy controls. This enhanced production ofinflammatory mediators (including but not limited to IL-1b, IL-4, IL-5,IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A and F, IL-18, IL-23, TNF-a,IFN-g, MIP family members, MCP-1, G- and GM-CSF, etc.) contributes tothe symptoms and pathology associated with IBDs such as Crohn's disease(CD) and ulcerative colitis (UC) by way of their effect(s) on activatinginflammatory pathways and downstream effector cells. These pathways andcomponents then lead to tissue and cell damage/destruction observed invivo. Therefore, this model can simulate this enhanced inflammatorymediator aspect of IBD. Furthermore, when intestinal tissue from healthycontrols or from human intestinal epithelial cell (IEC) lines iscultured in the presence of these inflammatory components, inflammatorypathway signaling can be observed, as well as evidence of tissue andcell damage.

Therapeutics that would be efficacious in human IBD in vivo would workin the above ex vivo or IEC models by inhibiting and/or neutralizing theproduction and/or presence of inflammatory mediators.

In this model, human intestinal tissue is collected from patients withIBD or from healthy controls undergoing intestinal biopsy, re-sectioningor from post-mortem tissue collection, and processed using amodification of Alexakis et al (Gut 53:85-90; 2004). Under asepticconditions, samples are gently cleaned with copious amounts of PBS,followed by culturing of minced sections of tissue, in the presence ofcomplete tissue culture media (plus antibiotics to prevent bacterialovergrowth). Samples from the same pool of minced tissue are treatedwith one of the following: vehicle (PBS); recombinant human (rh) IL-17A;rhIL-17F; or rhIL-17A+rhIL-17F. In addition, these are treated with orwithout an antibody of the invention (e.g. a cross-reactive orbispecific antibody). This experimental protocol is followed for studieswith human IEC lines, with the exception that cells are passaged fromexisting stocks. After varying times in culture (from 1 h to severaldays), supernatants are collected and analyzed for levels ofinflammatory mediators, including those listed above. In samples frompatients with IBD or in samples treated with rhIL-17A and/or F, levelsof inflammatory cytokines and chemokines are elevated compared tountreated healthy control tissue samples. The addition of an antibody ofthe invention markedly reduces the production of inflammatory mediators,and thus, would expect to be efficacious in human IBD.

Example 5 An Anti-IL-17A and IL-17F Antibody Decreases Disease Incidenceand Progression in a Multiple Sclerosis (MS) Model

Multiple sclerosis (MS) is a complex disease that is thought to bemediated by a number of factors, including the presence of lymphocyticand mononuclear cell inflammatory infiltrates and demyelinationthroughout the CNS. Microglia are macrophage-like cells that populatethe central nervous system (CNS) and become activated upon injury orinfection. Microglia have been implicated as playing critical roles invarious CNS diseases including MS, and may be used to study mechanism(s)of initiation, progression, and therapy of the disease (Nagai et al.Neurobiol Dis 8:1057-1068; 2001; Olson et al. Neurosci Methods128:33-43; 2003). Immortalized human microglial cell lines and/orestablished human astroglia cell lines can, therefore, be used to studysome of the effects of inflammatory mediators on these cell types andtheir potential for neutralization. Inflammatory mediators (includingbut not limited to IL-1b, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A andF, IL-18, IL-23, TNF-a, IFN-g, MIP family members, RANTES, IP-10, MCP-1,G- and GM-CSF, etc.) can contribute to the symptoms and pathologyassociated with MS by way of their effect(s) on activating inflammatorypathways and downstream effector cells.

In order to evaluate the pro-inflammatory actions of IL-17A and IL-17F,and the ability of an antibody of the invention to neutralize ordecrease these effects, cultured glial cells are treated with one of thefollowing: vehicle; rhIL-17A; rhIL-17F; rhIL-17A+IL-17F. In addition,these are treated with or without an antibody of the invention. Aftervarying times in culture (from 1 h to several days), supernatants andcells are collected and analyzed for levels and/or expression ofinflammatory mediators, including those listed above. Levels ofinflammatory cytokines and chemokines are elevated in the presence ofrhIL-17A and/or IL-17F compared to cultures treated with vehicle alone.The addition of antibodies of the present invention markedly reduces theproduction and expression of inflammatory mediators, and thus, wouldexpect to be efficacious in inflammatory aspects associated with humanMS.

Example 6 An Anti-IL-17A and IL-17F Antibody Decreases Disease Incidenceand Progression in a Rheumatoid Arthritis (RA) and Osteoarthritis (OA)Model

This model is designed to show that human synovial cultures (includingsynovial macrophages, synovial fibroblasts, and articular chondrocytes)and explants from patients with RA and OA produce higher levels ofinflammatory mediators compared to cultures/explants from healthycontrols. This enhanced production of inflammatory mediators (includingbut not limited to oncostatin M, IL-1b, IL-6, IL-8, IL-12, IL-15, IL-17A and F, IL-18, IL-23, TNF-a, IFN-g, IP-10, RANTES, RANKL, MIP familymembers, MCP-1, G- and GM-CSF, nitric oxide, etc.) contributes to thesymptoms and pathology associated with RA and OA by way of theireffect(s) on activating inflammatory pathways and downstream effectorcells. These pathways and components then lead to inflammatoryinfiltrates, cartilage and matrix loss/destruction, bone loss, andupregulation of prostaglandins and cyclooxygenases. Therefore, thismodel can simulate the destructive inflammatory aspects of RA and OA inin vitro and ex vivo experiments. Furthermore, when explants andsynovial cultures from healthy controls are cultured in the presence ofseveral of these inflammatory components (e.g. oncostatin M, TNF-a,IL-1b, IL-6, IL-17A and F, IL-15, etc.), inflammatory pathway signalingcan be observed. Therapeutics that would be efficacious in human RA invivo would work in the above in vitro and ex vivo models by inhibitingand/or neutralizing the production and/or presence of inflammatorymediators.

In this model, human synovial explants are collected from patients withRA, OA, or from healthy controls undergoing joint replacement or frompost-mortem tissue collection, and processed using a modification ofWooley and Tetlow (Arthritis Res 2: 65-70; 2000) and van 't H of et al(Rheumatology 39:1004-1008; 2000). Cultures of synovial fibroblasts,synovial macrophages and articular chondrocytes are also studied.Replicate samples are treated with one of the following: vehicle (PBS);recombinant human (rh) IL-17A; rhIL-17F; or rhIL-17A+rhIL-17F, and somesamples contain various combinations of oncostatin M, TNF-a, IL-1b,IL-6, IL-17A, IL-17F, and IL-15. In addition, these are treated with orwithout an antibody of the invention. After varying time of culture(from 1 h to several days), supernatants are collected and analyzed forlevels of inflammatory mediators, including those listed above. Insamples from patients with RA or OA, or in samples treated with rhIL-17Aand/or F (either alone or in combination with other inflammatorycytokines), levels of inflammatory cytokines and chemokines are elevatedcompared to untreated healthy control explants or in untreated cellcultures. The addition of antibodies of the present invention markedlyreduces the production of inflammatory mediators, and thus, would expectto be efficacious in human RA and OA.

Example 7 IL-17A and IL-17F Expression in Murine Disease Models

Four murine models of disease (asthma, DSS colitis, atopic dermatitisand experimental allergic encephalomyelitis) were analyzed using knowtechniques for the expression of IL-17A and IL-17F.

In the asthma model, IL-17A and IL-17F are expressed at very low toundetectable levels in lung, spleen, lung draining lymph nodes and lunginfiltrating cells in diseased and non-diseased mice.

Contrary to the asthma model, IL-17A and IL-17F were highly up-regulatedin diseased but not normal mice in the DSS-colitis model in bothproximal and distal colon. Neither cytokine was significantlyup-regulated in the mesenteric lymph node. Further, it was found thatup-regulation of both cytokines in the context of acute DSS-inducedcolitis and not in chronic DSS-induced colitis.

In atopic dermatitis, IL-17A mRNA was not detectable. IL-17F was foundto be expressed in both skin and skin-draining lymph nodes but did notappear to be significantly regulated with disease.

In experimental allergic encephalomyelitis, both IL-17A and IL-17Fappeared to up-regulated in spinal chord in diseased but not healthymice. IL-17F may have been more highly expressed in lymph nodes comparedto spinal cord but expression in the lymph nodes was not regulated withdisease. However, overall levels of expression in these tissues wasquite low.

In short, IL-17A and IL-17F expression appears to be regulated withdisease in the context of the DSS-induced colitis and experimentalallergic encephalomyelitis models but apparently not for asthma oratopic dermatitis.

Example 8 Construction of E. Coli Expression Vectors for Human IL-17Aand F IL-17A

Construction of pCHAN28

The human IL-17A expression construct was generated as follows. NativeIL-17A sequence were generated by PCR amplification with twooligonucleotide primers zc48,686 (SEQ ID NO:13) and zc48,685 (SEQ IDNO:14). The PCR conditions were as follows: 25 cycles at 94° C. forseconds, 50° C. for 30 seconds, and 72° C. for 1 minute; followed by a4° C. soak. The DNA fragment was precipitated with 2 volume absoluteethanol. Pellet was resuspended in 10 μL H₂O and used for recombinationinto SmaI cut recipient vector, pTAP238 to produce the constructsencoding human IL-17A. The resulting clones were designated as pCHAN28.They were digested with NotI (10 μL DNA, 5 μL buffer 3 New EnglandBioLabs, 2 μL Not I, 33 μL H₂O for 1 hour at 37° C.) and religated withT4 DNA ligase buffer (7 μL of the previous digest, 2-L of 5× buffer, 1μL of T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, tostreamline the vector. Aliquots of the DNA were digested with Pvu2 andPstI to confirm the absence of the yeast sequence. The human IL-17Aexpression constructs were transformed into E. coli strain W3110. Thepolynucleotide sequence for human IL-17A is shown in SEQ ID NO:5 and thecorresponding encoded IL-17A polypeptide is shown in SEQ ID NO:6.

IL-17F

Construction of pTAP419The human IL-17F expression construct was generated as follows. NativeIL-17F sequence were generated by PCR amplification with twooligonucleotide primers zc42,852 (SEQ ID NO:15) and zc42,854 (SEQ IDNO:16). The PCR conditions were as follows: 25 cycles at 94° C. for 30seconds, 50° C. for 30 seconds, and 72° C. for 1 minute; followed by a4° C. soak. The DNA fragment was precipitated with 2 volume absoluteethanol. Pellet was resuspended in 10 μL H₂O and used for recombinationinto SmaI cut recipient vector, pTAP238 to produce the constructsencoding human IL-17F. The resulting clones were designated as pTAP419.They were digested with NotI (10 μL DNA, 5 μL buffer 3 New EnglandBioLabs, 2 μL Not I, 33 μL H₂O for 1 hour at 37° C.) and religated withT4 DNA ligase buffer (7 mL of the previous digest, 2 μL of 5× buffer, 1L of T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, tostreamline the vector. Aliquots of the DNA were digested with Pvu2 andPstI to confirm the absence of the yeast sequence. The human IL-17Fexpression constructs were transformed into E. coli strain W3110. Thepolynucleotide sequence for human IL-17F is shown in SEQ ID NO:7, andthe corresponding encoded IL-17F polypeptide is shown in SEQ ID NO:8.

Example 9 Expression of IL-17A in E. coli

An expression plasmid containing pIL17A CH6 was constructed viahomologous recombination using human IL17A CH6 and the expression vectorpZMP20. The fragment was generated by PCR amplification using primerszc48895 (SEQ ID NO:17) and zc48893 (SEQ ID NO:18). The PCR fragmentIL17A CH6 contains the IL17A coding region fused to a 6×His tag on theC-terminus, which was made using human IL17A as the template. Thefragment includes a 5′ overlap with the pZMP20 vector sequence as wellas a 3′ overlap with the pZMP20 vector at the insertion point. PCRconditions used were as follows: 1 cycle, 94° C., 5 minutes; 35 cycles,94° C., 1 minute, followed by 55° C., 2 minutes, followed by 72° C., 3minutes; 1 cycle, 72° C., 10 minutes. The PCR reaction mixture was runon a 1% agarose gel and a band corresponding to the size of the insertwas gel-extracted using a QIAquick™ Gel Extraction Kit (Qiagen, Cat. No.28704).

Plasmid pZMP20 is a mammalian expression vector containing an expressioncassette having the CMV promoter, multiple restriction sites forinsertion of coding sequences, an otPA signal peptide sequence (removedvia recombination in this case); an internal ribosome entry site (IRES)element from poliovirus, and the extracellular domain of CD8 truncatedat the C-terminal end of the transmembrane domain; an E. coli origin ofreplication; a mammalian selectable marker expression unit comprising anSV40 promoter, enhancer and origin of replication, a DHFR gene, and theSV40 terminator; and URA3 and CEN-ARS sequences required for selectionand replication in S. cerevisiae. The plasmid pZMP20 was cut with BglII(creating the insertion point) prior to recombination in yeast with thePCR fragment. One hundred microliters of competent yeast (S. cerevisiae)cells were independently combined with 10 μl of the insert DNA and 100ng of cut pZMP20 vector, and the mix was transferred to a 0.2-cmelectroporation cuvette. The yeast/DNA mixture was electropulsed usingpower supply (BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV(5 kV/cm), ∞ ohms, and 25 μF. Six hundred μl of 1.2 M sorbitol was addedto the cuvette, and the yeast was plated in a 100-μl and 300 μl aliquotonto two URA-D plates and incubated at 30° C. After about 72 hours, theUra⁺ yeast transformants from a single plate were resuspended in 1 mlH₂O and spun briefly to pellet the yeast cells. The cell pellet wasresuspended in 0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mMNaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The five hundred microliters ofthe lysis mixture was added to an Eppendorf tube containing 250 μlacid-washed glass beads and 300 μl phenol-chloroform, was vortexed for 3minutes, and spun for 5 minutes in an Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase was transferred toa fresh tube, and the DNA was precipitated with 600 μl ethanol (EtOH),followed by centrifugation for 30 minutes at maximum speed. The tube wasdecanted and the pellet was washed with 1 mL of 70% ethanol. The tubewas decanted and the DNA pellet was resuspended in 30 μl TE.

Transformation of electrocompetent E. coli host cells (DH12S) was doneusing 5 μl of the yeast DNA prep and 50 μl of cells. The cells wereelectropulsed at 2.0 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was added and then the cells were plated in a 50 μl and a 200μl aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

The inserts of three clones for the construct was subjected to sequenceanalysis and one clone for each construct, containing the correctsequence, was selected. Larger scale plasmid DNA was isolated using acommercially available kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia,Calif.) according to manufacturer's instructions.

Expression of pIL17A CH6 was accomplished through transienttransfection. Six 1000 mL flasks were seeded with 250 mL of 293F cellsat 1E6 c/mL and were set aside. 20 mL of OptiMEM (Invitrogen, cat#31985-070) was placed in each of two 50 mL conical tubes. 2 mL ofLipofectamine 2000 (Invitrogen, cat #11668-019) was mixed into one ofthe OptiMEM containing 50 mL conical tubes and 1.5 mg of the IL17A CH6pZMP20 expression plasmid was placed in the other tube. The tubes wereinverted several times and allowed to incubate for 5 minutes at roomtemperature. The two tubes were then mixed together, inverted severaltimes, and allowed to incubate for 30 minutes at room temperature. TheDNA-Lipofectamine 2000 mixture was then evenly distributed into each ofthe six flasks while swirling the cell cultures. The flasks were thenplaced in an incubator on a shaker at 37° C., 6% CO₂, and shaking at 120RPM. The cultures were harvested 96 hours later.

Example 10 Expression of IL-17F in E. coli

An expression plasmid containing pIL17F CH6 was constructed viahomologous recombination using a human IL17F CH6 and the expressionvector pZMP20. The fragment was generated by PCR amplification usingprimers zc48894 (SEQ ID NO:19) and zc48892 (SEQ ID NO:20). The humanIL17F CH6 contains a the IL17F coding region fused to a 6×His tag on theC-terminus, which was made using a previously generated clone of humanIL17F as the template. The fragment includes a 5′ overlap with thepZMP20 vector sequence as well as a 3′ overlap with the pZMP20 vector atthe insertion point. PCR conditions used were as follows: 1 cycle, 94°C., 5 minutes; 35 cycles, 94° C., 1 minute, followed by 55° C., 2minutes, followed by 72° C., 3 minutes; 1 cycle, 72° C., 10 minutes. ThePCR reaction mixture was run on a 1% agarose gel and a bandcorresponding to the size of the insert was gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Cat. No. 28704).

Plasmid pZMP20 is a mammalian expression vector containing an expressioncassette having the CMV promoter, multiple restriction sites forinsertion of coding sequences, an otPA signal peptide sequence (removedvia recombination in this case); an internal ribosome entry site (IRES)element from poliovirus, and the extracellular domain of CD8 truncatedat the C-terminal end of the transmembrane domain; an E. coli origin ofreplication; a mammalian selectable marker expression unit comprising anSV40 promoter, enhancer and origin of replication, a DHFR gene, and theSV40 terminator; and URA3 and CEN-ARS sequences required for selectionand replication in S. cerevisiae.

The plasmid pZMP20 was cut with BglII (creating the insertion point)prior to recombination in yeast with the PCR fragment. One hundredmicroliters of competent yeast (S. cerevisiae) cells were independentlycombined with 10 μl of the insert DNA and 100 ng of cut pZMP20 vector,and the mix was transferred to a 0.2-cm electroporation cuvette. Theyeast/DNA mixture was electropulsed using power supply (BioRadLaboratories, Hercules, Calif.) settings of 0.75 kV (5 kV/cm), ∞ ohms,and 25 μF. Six hundred μl of 1.2 M sorbitol was added to the cuvette,and the yeast was plated in a 100-μl and 300 μl aliquot onto two URA-Dplates and incubated at 30° C. After about 72 hours, the Ura⁺ yeasttransformants from a single plate were resuspended in 1 ml H₂O and spunbriefly to pellet the yeast cells. The cell pellet was resuspended in0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). The five hundred microliters of the lysismixture was added to an Eppendorf tube containing 250 μl acid-washedglass beads and 300 μl phenol-chloroform, was vortexed for 3 minutes,and spun for 5 minutes in an Eppendorf centrifuge at maximum speed.Three hundred microliters of the aqueous phase was transferred to afresh tube, and the DNA was precipitated with 600 μl ethanol (EtOH),followed by centrifugation for 30 minutes at maximum speed. The tube wasdecanted and the pellet was washed with 1 mL of 70% ethanol. The tubewas decanted and the DNA pellet was resuspended in 30 μl TE.

Transformation of electrocompetent E. coli host cells (DH12S) was doneusing 5 μl of the yeast DNA prep and 50 μl of cells. The cells wereelectropulsed at 2.0 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was added and then the cells were plated in a 50 μl and a 200μl aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

The inserts of three clones for the construct was subjected to sequenceanalysis and one clone for each construct, containing the correctsequence, was selected. Larger scale plasmid DNA was isolated using acommercially available kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia,Calif.) according to manufacturer's instructions.

Expression of human IL17F CH6 was accomplished through transienttransfection. Six 1000 mL flasks were seeded with 250 mL of 293F cellsat 1E6 c/mL and were set aside. 20 mL of OptiMEM (Invitrogen, cat#31985-070) was placed in each of two 50 mL conical tubes. 2 mL ofLipofectamine 2000 (Invitrogen, cat #11668-019) was mixed into one ofthe OptiMEM containing 50 mL conical tubes and 1.5 mg of the IL17F CH6pZMP20 expression plasmid was placed in the other tube. The tubes wereinverted several times and allowed to incubate for 5 minutes at roomtemperature. The two tubes were then mixed together, inverted severaltimes, and allowed to incubate for 30 minutes at room temperature. TheDNA-Lipofectamine 2000 mixture was then evenly distributed into each ofthe six flasks while swirling the cell cultures. The flasks were thenplaced in an incubator on a shaker at 37° C., 6% CO₂, and shaking at 120RPM. The cultures were harvested 96 hours later.

Example 11 Characterization of Monoclonal Antibodies that Bind to BothIL-17A and IL-17F

Two assays are performed with the antibody containing supernatants fromthe best first round clones in each set. First, the concentration of IgGin each supernatant used in the neutralization assay is determined usingthe Mouse-IgG ELISA kit (catalog #1 333 151 (Roche Applied Science).This enables enabled a determination of specific neutralizing activityfor each supernatant and therefore identified hybridomas that wereproducing the most potent anti-IL17A and IL-17F and IL-17A plus IL-17Fneutralizing mAbs. From this analysis the most potent mAbs are selectedfor further characterization. Second, preliminary epitope specificity(“binning”) studies are performed with the supernatants using theBiacore 1000 surface plasmon resonance instrument.

Competitive Epitope Binding

Epitope binning and Western blotting experiments are performed to assessthe functional binding characteristics of the monoclonal antibodies toIL-17A and IL-17F. Binning studies are completed to determine antibodiesthat bind to different epitopes, or antigenic determinants, on IL-17A orIL-17F. Monoclonal antibodies that bind to the same, or a similar,epitope on IL-17A or IL-17F, respectively, are not able to bindsimultaneously and are functionally grouped into a single family or“bin”. Monoclonal antibodies that bind to different epitopes on IL-17Aor IL-17F, are able to bind simultaneously and are grouped into separatefamilies or “bins”. Experiments were performed using a Biacore 1000™instrument. Biacore is only one of a variety of assay formats that areroutinely used epitope bin panels of monoclonal antibodies. Manyreferences (e.g. The Epitope Mapping Protocols, Methods in MolecularBiology, Volume 6,6 Glenn E. Morris ed.) describe alternative methodsthat can be used (by those skilled in the art) to “bin” the monoclonalantibodies, and would be expected to provide consistent informationregarding the binding characteristics of the monoclonal antibodies toIL-17A and IL-17F. Epitope binning experiments are performed withsoluble, native antigen, E. coli derived or mammalian derivedrecombinant Il-17A and IL-17F.

Western Blotting

The ability of the monoclonal antibodies from the hybridomas to bind anddetect denatured and reduced/denatured IL-17A and/or IL-17F is alsoevaluated using a Western Blot format.

Epitope Binning A) Materials and Methods

Epitope binning studies are performed on a Biacore1000 ™ system(Biacore, Uppsalla Sweden). Methods are programmed using MethodDefinition Language (MDL) and run using Biacore Control Software, v 1.2.Polyclonal goat anti-Mouse IgG Fc antibody (Jackson ImmunoResearchLaboratories, West Grove, Pa.) is covalently immobilized to a BiacoreCM5 sensor chip and is used to bind (capture) the primary monoclonalantibody of test series to the chip. Unoccupied Fc binding sites on thechip are then blocked using a polyclonal IgG Fc fragment (JacksonImmunoResearch Laboratories, West Grove, Pa.). Subsequently, IL-17A orIL-17F is injected and allowed to specifically bind to the capturedprimary monoclonal antibody. The Biacore instrument measures the mass ofprotein bound to the sensor chip surface, and thus, binding of both theprimary antibody and IL-17A or IL-17F antigen, are verified for eachcycle. Following the binding of the primary antibody and antigen to thechip, a monoclonal antibody of the test series is injected as thesecondary antibody, and allowed to bind to the pre-bound antigen. If thesecondary monoclonal antibody is capable of binding the IL-17A or IL-17Fantigen simultaneously with the primary monoclonal antibody, an increasein mass on the surface of the chip, or binding, is detected. If,however, the secondary monoclonal antibody is not capable of binding theIL-17A or IL-17F antigen simultaneously with the primary monoclonalantibody, no additional mass, or binding, is detected. Each monoclonalantibody is tested against itself and is used as the negative control toestablish the level of the background (no-binding) signal.

Each purified monoclonal antibody is tested as the primary antibody incombination with the entire panel of selected monoclonal antibodies. Allpurified monoclonal antibodies are tested at equal concentrations. Inbetween cycles, the goat anti-Mouse IgG Fc capture antibody on the chipis regenerated with 20 mM HCl. Control cycles are run to demonstrate alack of response of the secondary antibody in the absence of primaryantibody or antigen. Data is compiled using BioEvaluation 3.2 RCIsoftware, then loaded into Excel™ for data processing.

B) Western Blotting

The ability of the monoclonal antibodies from each clone to detectdenatured and reduced/denatured Il-17A or IL-17F from two sources isassessed using a Western blot format. A rabbit polyclonal antibody knownto detect IL-17A and/or IL-17F in a Western blot format is used as apositive control.

Materials and Methods

The IL-17A and IL-17F antigen is obtained from two sources: IL-17A andIL-17F is either produced in E. coli or in mammalian cells such as 293cells (as described herein) and purified. Aliquots of each antigen (100ng/lane) is loaded onto 4-12% NuPAGE Bis-Tris gels (Invitrogen,Carlsbad, Calif.) in either non-reducing or reducing sample buffer(Invitrogen) along with molecular weight standards (SeeBlue;Invitrogen), and electrophoresis was performed in 1×MES running buffer(Invitrogen). Following electrophoresis, protein is transferred from thegel to 0.2 μm nitrocellulose membranes (Invitrogen). The nitrocelluloseblots are blocked overnight in 2.5% non-fat dried milk in Western Abuffer (ZymoGenetics, 50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.05%Igepal, 0.25% gelatin) then cut into sections and exposed to eachantibody (0.2 μg/mL of each monoclonal or 0.5 μg/mL of the rabbitpolyclonal antibody in Western A buffer). The blots are then probed witha secondary antibody conjugated to horseradish peroxidase; sheepanti-mouse IgG-HRP (Amersham: Piscataway, N.J.) for the monoclonalantibodies and donkey anti-rabbit Ig-HRP (Amersham) for the polyclonalantibodies. Bound antibody is then detected using a chemiluminescentreagent (Lumi-Light Plus Reagent: Roche, Mannheim, Germany) and imagesof the blots are recorded on a Lumi-Imager (Mannheim-Boehringer) orX-ray film (Kodak).

Example 12 Purification of C-Terminally his Tagged IL17A Protein from293 Transient Cell System Murine IL-17A

Delivered media adjusted to 25 mM Imidazole, 500 mM NaCl pH 7.5, viaaddition of solid (Fluka and JT Baker, respectively) (1.41 L totalvolume). Expression of his-tagged target analyzed via western blot viaRP-HPLC (1.11 mg/L). Adjusted media loaded over Ni NTA His BindSuperflow (Novagen) column (5 mL, 1 cm diameter, Millipore) overnight at4° C. Flow through checked via RP-HPLC and Western blot to be devoid ofIL17A target. Ni NTA column washed with 50 mM NaPO4, 25 mM Imidazole,0.5M NaCl pH 7.5 until UV @ A280 nm baseline stabilized. Column elutedin two steps: buffer as above adjusted to 45 mM and 500 mM Imidazoleusing 500 mM Imidazole stock. Elution fractions checked by silver stainanalysis, with those containing target pooled (500 mM step elution). NiNTA pool analyzed via RP-HPLC. Ni NTA pool concentrated to 2 mL against10 kD MWCO Ultracel membrane (Millipore) and injected over Superdex® 75column (GE Healthcare, 12/60 mm) running in 50 mM NaPO4, 109 mM NaCl pH7.3 at 1.02 mL/min. Two peaks resolved and analyzed via silver stain.The murine IL17A resolved nicely from the remaining contaminatingproteins. Fractions containing pure target pooled, concentrated again to2.0 mL using 10 kDa MWCO Ultracel membrane (Millipore), 0.22 umfiltered, and aliquoted.

Human IL-17A

Delivered media adjusted to 25 mM Imidazole, 500 mM NaCl pH 7.5, viaaddition of solid (Fluka and J T Baker, respectively) (1.41 L totalvolume). Expression of his-tagged target analyzed via western blot usingA1022G as standard and also via RP-HPLC (3.19 mg/L). Adjusted medialoaded over Ni NTA His Bind Superflow (Novagen) column (5 mL, 1 cmdiameter, Millipore) overnight at 4° C. Flow through checked via RP-HPLCand Western blot to be devoid of IL17A target. Ni NTA column washed with50 mM NaPO4, 25 mM Imidazole, 0.5M NaCl pH 7.5 until UV @ A280 nmbaseline stabilized. Column eluted in two steps: buffer as aboveadjusted to 45 mM and 500 mM Imidazole using 500 mM Imidazole stock.Elution fractions checked by silver stain analysis, with thosecontaining target pooled (500 mM step elution). Ni NTA pool analyzed viaRP-HPLC using A1022F as standard. Ni NTA pool concentrated to 5 mLagainst 10 kD MWCO Ultracel membrane (Millipore) and injected overSuperdex® 75 column (GE Healthcare, 26/60 mm) running in 50 mM NaPO4,109 mM NaCl pH 7.3 at 2.71 mL/min. Two peaks resolved and analyzed viasilver stain. The murine IL17A resolved nicely from the remainingcontaminating proteins. Fractions containing pure target pooled,concentrated again to 9.0 mL using 10 kDa MWCO Ultracel membrane(Millipore), 0.22 um filtered, and aliquoted.

Example 13 Purification of C-Terminally his Tagged IL17F Protein from293 Transient Cell System

IMAC affinity capture on 293F media from transient expression. Mediaadjusted to 25 mM Imidazole; 25 mM NaPhos; 400 mM NaCl and pH 7.5. Thusadjusted media was loaded at 1 ml/min over a 5 ml bed of Qiagen NTASuperflow (1 cm dia) equilibrated in 25 mM NaPhos; 25 mM Imidazole; 500mM NaCl at pH 7.5. Upon completing the load, the column was washed with20 CV equilibration buffer before eluting with a 100 CV gradient formedbetween elution buffer and 25 mM NaPhos; 500 mM Imidazole; 500 mM NaClat pH 7.5. Fractions were assayed by RP-HPLC for product, pooled andconcentrated for SEC step. The concentrated pool from the IMAC step wasinjected onto a Pharmacia Superdex 75 SEC column equilibrated in 50 mMNaPhos; 109 mM NaCl at pH 7.2. A major symmetric peak eluting at −0.5 CVcontains the product. Samples were pooled and sterile filtered to 0.2micron in preparation for aliquoting.

Example 14 Efficacy of an Antibody that Binds Both IL-17A and IL-17F inHuman IBD Samples Via Epithelial Barrier Function

Maintenance of epithelial barrier integrity is a critical factor in thepreservation of a healthy gastrointestinal tract. Experimental evidencesuggests that leakiness of the epithelial barrier in the gut maycontribute to the development of IBD. Immune cells located in theintestinal lamina propria generally interact with intestinal epithelialcells via cell to cell contact or production of soluble factors tomaintain immune surveillance and contribute to epithelial barrierintegrity. However, prolonged or dysregulated immune-mediatedinflammation may contribute to defects in epithelial barrier cellintegrity and function. The following study is designed to measure thedirect effect(s) of T cell-derived IL-17A and/or IL-17F on epithelialbarrier integrity.

In this example, intestinal epithelial cell lines, like Caco-2 cells,are differentiated on semipermeable membranes and co-cultured on thebasolateral side with either T cells or monocytes derived from biopsiesfrom IBD patients or normal individuals. Epithelial monolayer integrityis monitored over time using assessment of transepithelial electricalresistance or resistance of the monolayer to dye diffusion. Decreases intransepithial resistance of monolayers in co-cultures would suggest adisruption in the monolayer induced by the activity of the T cells ormonocytes in the co-culture. Inhibitors of IL-17A and IL-17F such as theantibodies of the present invention could be used to determine therelative contribution of IL-17A and IL-17F to the disruption of theepithelial monolayer and test whether inhibitors of IL-17A and IL-17Fwould be effective in maintaining epithelial barrier integrity.Prevention of epithelial monolayer disruption induced by activated Tcells by such molecules would suggest that the antibodies of the presentinvention may be effective for the therapeutic treatment of IBD inhumans.

Co-culture systems could also be generated using monolayers formed byprimary epithelium from IBD patients to determine whether these cellsare more sensitive to IL-17A and IL-17F compared to epithelial cellsderived from healthy individuals. If so, these data would suggest thatinhibiting IL-17A and IL-17F would be a suitable strategy for thetherapeutic treatment of IBD.

Example 15 Effects of IL-17A and IL-17F on Lamina PropPria T Cells andMonocytes/Macrophages from Normal and Human IBD Samples

Dysregulated or sustained immune-mediated inflammation may contribute tothe symptoms and pathology associated with IBD by way of tissue damageor permanent skewing to inappropriate or prolonged immune responses.This model can determine the potential down-stream consequences ofexposure of disease-associated T cells and monocytes to IL-17A andIL-17F which may be present in the immediate environmental cytokinemileu of the intestinal tissue.

Therapeutics that would be efficacious in human IBD in vivo would workin the above ex vivo models by inhibiting and/or neutralizing theproduction and/or presence of inflammatory mediators (including but notlimited to IL-1b, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 Aand F, IL-18, IL-23, TNF-a, IFN-g, MIP family members, MCP-1, G- andGM-CSF, etc.).

In this model, T cells and monocytes/macrophages are isolated frombiopsy samples by carefully mincing biopsies with scissors in HBSS,treating with collagenase and Dispase II and incubating for 1 hr at 37°C. in a shaker. The cell suspension is filtered through nylon mesh toremove debris and cell clumps and washed multiple times in HBSS. T cellsand macrophage/monocytes can be isolated using direct cell sorting orbead-depletion/enrichment protocols. Isolated cells are incubated in thepresence of IL-17A and IL-17F. This induces the production ofinflammatory mediators by T cells and monocytes/macrophages or resultsin skewing subsequent T cell responses to highly pro-inflammatoryresponses. Comparisons between the types of inflammatory mediatorsproduced by cells from IBD patients and those from cells of normalindividuals can be made and might suggest that T cells andmonocyte/macrophages from IBD patients produce a more pro-inflammatoryprofile in the presence of IL-17A and IL-17F. The addition of anantibody of the present invention to neutralize the production ofdownstream inflammatory mediators induced by IL-17A and IL-17F suggeststhat such antibodies may be efficacious in the therapeutic treatment ofpatients with IBD.

Example 16 Efficacy of Antibodies that to Both IL-17A and IL-17F inIrritable Bowl Syndrome (“IBS”): CNS-Directed Pathogenesis

A model focusing on, primary CNS-directed pathogenesis of IBS whichemploys stress stimuli to induce symptoms characteristic of IBS. Theneonatal psychosocial stress model mimics some clinical featuresassociated with IBS patients including visceral hyperalgesia, diarrheaand stress-sensitivity. Daily separation of the litter from theirmothers for 180 minutes each day during postnatal days 4-18 will resultin an alteration of maternal behavior and significantly reduce times ofthe licking/grooming behavior. The stress on the neonates results inpermanent changes in the CNS resulting in altered stress-inducedvisceral and somatic pain sensitivity. Colonic motor function inresponse to stress is enhanced in these animals and preliminary datashows evidence of increased intestinal permeability (Mayer et al.,2002). Treatment with an antibody of the present invention andsubsequent analysis of colonic motor function, epithelial permeabilityand response to stress stimuli could determine efficacy in this animalmodel of IBS. Decreases in the incidence of symptoms following treatmentwith these inhibitors would suggest potential efficacy in the treatmentof IBS.

Example 17 Efficacy of Antibodies that to Both IL-17A and IL-17F inIrritable Bowl Syndrome (“IBS”): Primary Gut-Directed Inducers of Stress

This is a model focusing on primary gut-directed inducers of stress(i.e., gut inflammation, infection or physical stress). Animal studieshave indicated that low-grade inflammation or immune activation may be abasis for altered motility, and/or afferent and epithelial function ofthe gut (Mayer et al., 2002). In this model, daily colon irritation isproduced in neonatal animals (days 8-21) in the form of dailyintracolonic injection of mustard oil. Mustard oil is a neural stimulantand has been shown to induce visceral hyperalgesia followingintracolonic administration. This model mimics key features of the IBSincluding visceral hypersensitivity and alteration in bowel habits.Animals also present with diarrhea or constipation, a key feature of IBSpatients (Mayer et al., 2002; Kimball et al., 2005). An antibody of thepresent invention could be delivered to determine changes in thedevelopment of symptoms associated with this model. Decreases in theincidence or magnitude of visceral hypersensitivity and altered gutmotility following therapeutic treatment with our inhibitors wouldsuggest a potential for these molecules to be efficacious in thetreatment of IBS.

Example 18 Generation of the IL-17A/F Antibodies A) Capture Assay

The ability of anti-human IL-17F or anti-human IL-17A antibodies in theantisera to bind to IL-17F and/or IL-17A was assessed using a captureELISA assay. In this assay, wells of 96 well polystyrene ELISA plateswere first coated with 100 μL/well of goat anti-mouse IgG, Fc specificantibody (Jackson Immunoresearch) at a concentration of 1000 ng/mL inCoating Buffer (0.1M Na₂CO₃, pH 9.6). One plate for each ligand wasprepared. Plates were incubated overnight at 4° C. after which unboundantibody was aspirated and the plates washed twice with 300 μL/well ofWash Buffer (PBS-Tween defined as 0.137M NaCl, 0.0027M KCl, 0.0072MNa₂HPO₄, 0.0015M KH₂PO₄, 0.05% v/v polysorbate 20, pH 7.2). Wells wereblocked with 200 μL/well of Blocking Buffer (PBS-Tween plus 1% w/vbovine serum albumin (BSA)) for 1 hour, after which the plates werewashed twice with Wash Buffer. Serial 10-fold dilutions (in BlockingBuffer) of the sera were prepared beginning with an initial dilution of1:1000 and ranged to 1:1,000,000. Duplicate samples of each dilutionwere then transferred to the assay plate, 100 L/well, in order to bindmouse IgG in the sera to the assay plate through the Fc portion of themolecule. Normal mouse sera served as a negative control, humanIL17RC-Fc protein was added as a positive assay control. NOTE: Coatingfor this control was goat anti-human IgG, Fc specific antibody (JacksonImmunoresearch). Following a 1-hour incubation at RT, the wells wereaspirated and the plates washed twice as described above. BiotinylatedIL17F (6:1 molar ratio of biotin:protein) or biotinylated IL17A (10:1molar ratio of biotin:protein) at concentrations of 500 ng/mL were thenadded to the wells (separate plates), 100 μL/well. Following a 1-hourincubation at RT, unbound biotinylated ligand was aspirated from thewells and the plates washed twice. Horseradish peroxidase labeledstreptavidin (Pierce, Rockford, Ill.) at a concentration of 500 ng/mLwas then added to each well, 100 μL/well, and the plates incubated at RTfor 1 hour. After removal of unbound HRP-SA, the plates were washed 2times, 100 μL/well of tetra methyl benzidine (TMB) (BioFX Laboratories,Owings Mills, Md.) added to each well and the plates incubated for 2minutes at RT. Color development was stopped by the addition of 100μL/well of 450 nm TMB Stop Reagent (BioFX Laboratories, Owings Mills,Md.) and the absorbance values of the wells read on a Molecular DevicesSpectra MAX 340 instrument at 450 nm.

B) Direct Assay

The ability of anti-human IL-17F or anti-human IL-17A antibodies in theantisera to bind to IL-17F and/or IL-17A was assessed using a directELISA assay. In this assay, wells of 96 well polystyrene ELISA plateswere first coated with 100 μL/well of IL-17F or IL-17A at concentrationsof 1000 ng/mL in Coating Buffer (0.1M Na₂CO₃, pH 9.6). One plate foreach ligand was prepared. Plates were incubated overnight at 4° C. afterwhich unbound protein was aspirated and the plates washed twice with 300μL/well of Wash Buffer (PBS-Tween defined as 0.137M NaCl, 0.0027M KCl,0.0072M Na₂HPO₄, 0.0015M KH₂PO₄, 0.05% v/v polysorbate 20, pH 7.2).Wells were blocked with 200 μL/well of Blocking Buffer (PBS-Tween plus1% w/v bovine serum albumin (BSA)) for 1 hour, after which the plateswere washed twice with Wash Buffer. Serial 10-fold dilutions (inBlocking Buffer) of the sera were prepared beginning with an initialdilution of 1:1000 and ranged to 1:1,000,000. Duplicate samples of eachdilution were then transferred to the assay plate, 100 μL/well, in orderto bind specific protein in the sera to the assay plate. Normal mousesera served as a negative control, zcytor14 (lot A1034F) was added as apositive assay control. Following a 1-hour incubation at RT, the wellswere aspirated and the plates washed twice as described above.Horseradish peroxidase labeled goat anti-mouse IgG, Fc specific antibody(Jackson Immunoresearch) at a concentration of 1:5000 was then added tothe wells both plates, 100 μL/well. Following a 1-hour incubation at RT,unbound antibody was aspirated from the wells and the plates washedtwice. Tetra methyl benzidine (TMB) (BioFX Laboratories, Owings Mills,Md.), 100 μL/well, was added to each well and the plates incubated for 2minutes at RT. Color development was stopped by the addition of 100μL/well of 450 nm. TMB Stop Reagent (BioFX Laboratories, Owings Mills,Md.) and the absorbance values of the wells read on a Molecular DevicesSpectra MAX 340 instrument at 450 nm.

C) Neutralization Assay

The ability of anti-human IL-17F or anti-human IL-17A antibodies in theantisera to inhibit (neutralize) the stimulatory activity of IL-17Fand/or IL-17A through its cognate receptor was assessed using a platebased neutralization assay. In this assay, wells of 96 well polystyreneELISA plates were first coated with 100 mL/well of human IL17RC-Fcprotein at a concentration of 1000 ng/mL in Coating Buffer (0.1M Na₂CO₃,pH 9.6). One plate for each ligand was prepared. Plates were incubatedovernight at 4° C. after which unbound receptor was aspirated and theplates washed twice with 300 μL/well of Wash Buffer (PBS-Tween definedas 0.137M NaCl, 0.0027M KCl, 0.0072M Na₂HPO₄, 0.0015M KH₂PO₄, 0.05% v/vpolysorbate 20, pH 7.2). Wells were blocked with 200 μL/well of BlockingBuffer (PBS-Tween plus 1% w/v bovine serum albumin (BSA)) for 1 hour,after which the plates were washed twice with Wash Buffer. Serial10-fold dilutions (in Blocking Buffer) of the sera were preparedbeginning with an initial dilution of 1:500 and ranged to 1:500,000.Biotinylated IL-17F (6:1 molar ratio of biotin:protein) or biotinylatedIL-17A (10:1 molar ratio of biotin:protein) at a concentrations of 200ng/ml were then added to the wells of the dilution plates (separateplates), 100 μL/well, mixed well by pipetting up and down several timesand incubated 1 hour at RT. NOTE: The mixing of the sera dilutions andthe biotinylated ligands at equal volumes results in the dilution seriesbecoming 1:1000 through 1:1,000,000 and the ligand concentrationsbecoming 100 ng/ml. Duplicate samples of each sera dilution/biotinylatedligand solution were then transferred to the assay plate, 100 μL/well.Normal mouse sera served as a negative control, human IL-17RC-Fc proteinwas added as a positive assay control. Following a 1-hour incubation atRT, the wells were aspirated and the plates washed twice as describedabove. Horseradish peroxidase labeled streptavidin (Pierce, Rockford,Ill.) at a concentration of 500 ng/mL was then added to each well, 100μL/well, and the plates incubated at RT for 1 hour. After removal ofunbound HRP-SA, the plates were washed 2 times, 100 μL/well of tetramethyl benzidine (TMB) (BioFX Laboratories, Owings Mills, Md.) added toeach well and the plates incubated for 3 minutes at RT, Colordevelopment was stopped by the addition of 100 μL/well of 450 nm TMBStop Reagent (BioFX Laboratories, Owings Mills, Md.) and the absorbancevalues of the wells read on a Molecular Devices Spectra MAX 340instrument at 450 nm.

D) Immunization Scheme

Five balb/C mice were immunized with IL-17F-BSA (50 μg each) every twoweeks for 6 weeks (3 immunizations) via interperitoneal injection. Twoweeks later, these mice were boosted with IL17A-BSA (50 μg each) viainterperitoneal injection. Bleeds were taken each week after the last 3boosts for sera evaluation. Approximately two months after the lastboost, all 5 animals were boosted with IL-17A-BSA (50 μg each) viasub-cutaneous injection and a final bleed taken for sera evaluation.

E) Conclusion of Sera Evaluation by ELISA

Both the capture ELISA assay as well as the direct ELISA assay indicatethat all five mice developed a significant antibody response to IL-17F.The direct assay indicates that four of the five mice also moderatelybinds IL-17A and one mouse weakly binds IL-17A. The neutralization assayindicates that two of the five mice moderately inhibit binding of IL-17Fand two others weakly inhibit binding of IL-17F. One mouse does notinhibit binding of IL-17F at all. Also indicated by this assay is thattwo of the five mice weakly inhibit binding of IL-17A, whereas the otherthree do not inhibit binding. One mouse inhibits binding of both ligandsto different degrees.

F) Fusion Procedure

After a minimum of four weeks post final immunization, the mouse withthe most significant IL-17F and IL-17A neutralization titer wasimmunized a final time with approximately 50 μg of IL-17A-BSA in PBS viasub-cutaneous injection. Under normal conditions, five days later thespleen and lymph nodes of this mouse would have been harvested, preparedinto a single cell suspension and fused to the Ag8 mouse myeloma cellline at a 2:1 lymphoid cell:myeloma cell ratio with PEG 1500 using thestandard in-house protocol. In this instance, the mouse died postinjection and the spleen was harvested, prepared into a single cellsuspension and frozen at −80° C. for 5 days. After quickly thawing thespleen cell suspension, the fusion was completed as stated above. Thefusion mixture was distributed into a series of 96 well flat-bottomedplates. Wells of the fusion plates were fed on days 4-7 (minimum oftwice, maximum of 3 times). Wells were assayed eight days after platingof the fusion.

G) Screening of the Fusion

The capture ELISA and neutralization ELISA for IL-17F and IL-17A asdescribed above were used to screen except that hybridoma supernatantswere tested undiluted from the culture plates. All ‘positive’ clones(described in detail in Example 19) were verified by repeating bothassays with the samples in duplicate. All ‘positive’ clones wereexpanded into culture in 24 well plates. When the density of the 24 wellcultures was approximately 4-6×10⁵ cells/mL, the supernatants(approximately 1.8 mL) were individually collected and stored for eachclone and the cells from each well cryopreserved. The collectedsupernatants were used to further evaluate which clones meet therequested reagent needs. The appropriate clones were subjected to 1^(st)and 2^(nd) round cloning prior to scale up for purification. Hybridomasexpressing monoclonal antibodies to IL-17A and IL-17F were depositedwith the American Type Tissue Culture Collection (ATCC; Manassas Va.)patent depository as original deposits under the Budapest Treaty andwere given the following ATCC Accession Nos.: clone 339.15.5.3 (ATCCPatent Deposit Designation PTA-7987, deposited on Nov. 7, 2006) clone3391536 ATCC Patent Deposit Designation PTA-7988 deposited on Nov. 7,2006); clone 339.15.3.6 (ATCC Patent Deposit Designation PTA-7988,deposited on Nov. 7, 2006); and clone 339.15.6.16 (ATCC Patent DepositDesignation PTA-7989, deposited on Nov. 7, 2006.

Example 19 IL-17A/F mAb Competitive Binding Assay Protocol

To assess the ability of the IL-17A/F antibodies of the presentinvention (as disclosed in Example 18) to bind the ligands IL-17A andIL-17F, a Flow Cytometry-based competitive binding assay was utilized.Incubation of a BHK cell line stably transfected with full lengthIL-17RC in the presence of the ligands IL-17A or IL-17F, and an IL-17A/Fantibody of the present invention targeted to bind the ligands allowsfor detection and relative quantification of ligand bound to the cellsurface (and therefore unbound by the antibody). The biotinylation ofthe ligand allows for FACS detection using a secondary Streptavidinconjugated fluorophore. A reduction in cell bound ligand over atitration of the antibody is recorded as a reduction in the meanfluorescence of the cells.

Biotinylated ligands are individually pre-mixed at 1 ug/ml withtitrating amounts of antibody in staining media (HBSS+1% BSA+0.1%NaAzide+10 mM HEPES) in 100 ul volumes and incubated at RT for 15minutes. A BHK cell line stably transfected with full length IL17RC isprepared for ligand staining by resuspension with Versene (Invitrogencat. 15040-066), equilibrating to 2×10e5 cells/100 ul, pelleting, andresuspension in the ligand/antibody pre-mix. Stained cells are incubatedat 4° for 30 minutes, washed 1× in staining media, and stained withStreptavidin-PE (BD Pharmingen cat. 554061) at a 1:100 ratio. Cells areincubated at 4° in the dark for 30 minutes, washed 2× in staining media,and re-suspended in a 1:1 ratio of staining media and Cytofix (BDBioscience 554655). The BD LSRII Flow Cytometer or similar instrument isused for data collection and analysis. The graph as shown in FIGURErepresents a typical assay result using the proceeding protocol. Thegraph was generated using the Prizm software program. The Y valuesrepresent the MFI normalized to maximum and minimum (100% and 0%) basedon ligand only and no ligand/no antibody control wells, and thus thepercent binding of the ligand to the cells. The software calculates theIC50 for each curve.

Table 1 contains the IC₅₀ values obtained for each IL-17A/F antibody.

TABLE 1 Competitive Binding IC50 (ug/mL) Clone Reactivity IL17A IL17F339.15.3.6 IL17A & F 38.0 3.5 339.15.5.3 IL17A & F 35.0 3.6 339.15.6.16IL17A & F 28.0 3.5

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

What is claimed is:
 1. An isolated, cross-reactive monoclonal antibodyor antigen-binding fragment thereof that binds to both IL-17A (SEQ IDNO:2) and IL-17F (SEQ ID NO:4), wherein said antibody binds to adiscontinuous IL-17F epitope comprising amino acid residues 105-109 and147-152 of SEQ ID NO:4 and a discontinuous IL-17A epitope comprisingamino acid residues 107-111 and 149-154 of SEQ ID NO:2; and wherein saidantibody reduces the pro-inflammatory activity of both IL-17A andIL-17F.
 2. The antibody of claim 1, wherein the antibody is selectedfrom the group consisting of a murine antibody, a chimeric antibody, ahumanized antibody, and a human antibody.
 3. The antibody of claim 1,wherein the antigen-binding fragment is a single chain antibody.
 4. Theantibody of claim 1, wherein the antigen-binding fragment is selectedfrom the group consisting of Fab, Fab′, F(ab′)₂, and Fv antibodyfragments.
 5. A composition comprising: the antibody or antigen-bindingfragment of claim 1; and a pharmaceutically acceptable vehicle, carrier,or excipient.